Modulation of synaptic maintenance

ABSTRACT

C1q is shown to be expressed in neurons, where it acts as a signal for synapse elimination. Methods are provided for protecting or treating an individual suffering from adverse effects of synapse loss. These findings have broad implications for a variety of clinical conditions, including treating and preventing neurodegenerative diseases such as Alzheimer&#39;s disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority under 35U.S.C. § 120 to U.S. Application, U.S. Ser. No. 13/586,556, filed Aug.15, 2012, which claims priority under 35 U.S.C. § 119(e) to U.S.provisional application, U.S. Ser. No. 61/523,702, filed Aug. 15, 2011,and is also a continuation-in-part of and claims priority under 35U.S.C. § 120 to U.S. Application, U.S. Ser. No. 13/326,180, filed Dec.14, 2011, granted as U.S. Pat. No. 9,149,444, which is a continuation ofand claims priority under 35 U.S.C. § 120 to U.S. Application, U.S. Ser.No. 11/636,001, filed Dec. 8, 2006, granted as U.S. Pat. No. 8,148,330,which claims priority under 35 U.S.C. § 119(e) to U.S. provisionalapplication, U.S. Ser. No. 60/749,071, filed on Dec. 9, 2005, each ofwhich is incorporated herein by reference.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under contracts EY011310and DA015043 awarded by the National Institutes of Health The Governmenthas certain rights in this invention.

BACKGROUND

The formation of precise neuronal circuits during development is ahighly regulated and dynamic process. Excess numbers of synapses arefirst generated to establish the initial wiring pattern of the brain,but the formation of mature, precise neuronal circuits requires theselective elimination and pruning of specific synapses. Neuronalactivity plays a critical role in this refinement phase, butsurprisingly, the specific molecular mechanisms underlying synapseelimination remain a mystery. In the adult brain, synapse loss oftenoccurs long before the pathology and clinical symptoms in manyneurodegenerative diseases. Identification of the instructivemolecule(s) that mark synapses for elimination during development couldalso provide important clinical insight about therapeutic targets fordevastating diseases such as Alzheimer's.

Synapses are specialized cell adhesions that are the fundamentalfunctional units of the nervous system, and they are generated duringdevelopment with amazing precision and fidelity. During synaptogenesis,synapses form, mature, and stabilize and are also eliminated by aprocess that requires intimate communication between pre- andpostsynaptic partners. In addition, there may be environmentaldeterminants that help to control the timing, location, and number ofsynapses.

Synapses occur between neuron and neuron and, in the periphery, betweenneuron and effector cell, e.g. muscle. Functional contact between twoneurons may occur between axon and cell body, axon and dendrite, cellbody and cell body, or dendrite and dendrite. It is this functionalcontact that allows neurotransmission. Many neurologic and psychiatricdiseases are caused by pathologic overactivity or underactivity ofneurotransmission; and many drugs can modify neurotransmission, forexamples hallucinogens and antipsychotic drugs.

Glial cells associated with synapses, either astrocytes in the CNS orSchwann cells in the PNS, are thought to provide synaptic insulation bypreventing neurotransmitter spillover to neighboring synapses and theyalso help to terminate neurotransmitter action. In addition, glial cellssupply synapses with energetic substrates. The possible requirement ofglia for synapse formation is suggested by the temporal association ofsynaptic development with glial development: although most neurons areborn prior to the birth of most glia, the vast majority of synapsesdevelop during the first few weeks of postnatal life, during the periodof glial generation. For example, axons of retinal ganglion cells (RGCs)reach their target in the superior colliculus by embryonic day 16, butthey do not form many synapses until the second postnatal week,coinciding with glial generation. Therefore the formation of many ormost synapses is delayed until glial cells are present.

During development, competition between axons causes permanent removalof synaptic connections. The synapses to be eliminated becomeprogressively weaker, are eliminated, and then the competing axonextends axonal processes to occupy those sites. These findings have leadto a simple model in which synaptic transmission produces twopostsynaptic signals: a short range protective signal and a longer rangeelimination (punishment) signal. Functionally weak synapses are notprotected from the elimination signal of neighboring stronger synapses,resulting in the disappearance of postsynaptic receptors and withdrawalof the axon. This withdrawal then provides the opportunity for thestronger axon to expand into the vacated territory. The identity of thepunishment and protection signals have heretofore been unknown.

Shortly after birth, neonatal brains undergo a period of intensesynaptic proliferation to levels far greater than those seen in adultbrains. Later in infancy there is a spontaneous, normal period ofsynaptic pruning or reduction. In rhesus monkeys the synaptic density(i.e., the number of synapses per unit of brain tissue volume) peaks at2 to 4 months of age and then gradually declines until about age 3years, where it remains at adult levels. The proliferation and pruningappear to occur uniformly throughout the rhesus cortex.

Data on human brains suggest that these programmed fluctuations insynaptic density also occur, but they vary by brain region.Synaptogenesis in the visual cortex, for example, begins its rapidgrowth at about age 2 months, peaks at 8 to 10 months, and then declinesgradually until about age 10 years. By contrast, synaptogenesis in thefrontal cortex begins and peaks later, and pruning is not complete untiladolescence. Interpretation of these findings about synaptic densitycounts is further complicated because synaptogenesis and pruning mayoccur at different rates in different structures within the same brainregion or even for a particular group of neurons in different parts oftheir dendritic fields.

Two phenomena thought to be related to this process of synaptogenesisand pruning are those of so-called “critical periods” and neuralplasticity, both of which have been studied extensively over the past 30years. Deprivation of adequate sensory or motor input during particulartimes in a specific brain system's development (i.e., the criticalperiod) can lead to impairments in that system's functioning, both atthat time and in the future.

It is now thought that the need for appropriate sensory input isgreatest during a brain system's period of rapid synaptogenesis and thatexperiential input helps shape the particular synaptic connections thatare formed and also which ones are eliminated. This process correspondsto the “experience-expectant” type of neural plasticity that is tied tothe brain's developmental timetable. By contrast, “experience-dependent”plasticity allows incorporation of useful but idiosyncratic informationthroughout life. The onset of critical periods and their durations varywidely over the different neural systems in the brain. At present, it isnot known whether there are critical periods during which particulartypes of stimulation are needed and after which plasticity is greatlyreduced. Nor is it known whether neuronal plasticity responsiveness ispresent in discreet, sensitive periods versus demonstrating more gradualdecrease over time.

Although there are varied etiologies among neurodegenerative diseases,one cellular commonality which exists among all neurons is the synapse.Degeneration of functional synapses is of crucial importance tounderstanding the primary mechanisms of overall neurodegeneration.Evidence suggests that synapse loss precedes neuron loss, e.g. in earlyAlzheimer's Disease (AD). Several studies have correlated synapse losswith clinically defined neurological impairment. For example,statistical analysis has shown that synapse loss is more closelycorrelated with cognitive impairment in AD than are plaques and tangles.Moreover, a variety of proteins found in pathological hallmarks ofneurodegenerative diseases are synaptic proteins or cleavage products ofsynaptic proteins. These include APP, amyloid precursor protein,alpha-synuclein, the precursor of NAC peptide found in Lewy bodies inParkinson's Disease, and PrP.

These observations emphasize that synapse loss is a central event inneurodegeneration and that synaptic proteins have been involved in theneuropathology of disease. Despite this fundamental understanding, therehas been little systematic study of synapse loss or the role of synapticproteins associated with pathology. The modulation of synapsemaintenance and loss is of great interest for the treatment of a varietyof nervous system disorders. The present invention addresses this issue.

SUMMARY OF THE INVENTION

Methods are provided for the modulation of synaptic development,including synapse elimination. It has been found that specificcomplement proteins are expressed by neurons, and are involved in thepathway for elimination of synapses. Agents that inhibit complementactivation, including agents that block specific components, such asC1q, can prevent synapse elimination from neurons. Neurons affected bysynapse loss may be central nervous system neurons, or peripheralnervous system neurons.

In some aspects of the invention, methods are provided for protecting ortreating an individual from synapse loss, e.g. an individual sufferingfrom adverse effects of synapse loss, or an individual at risk ofsuffering from the adverse effects of synapse loss. These findings havebroad implications for a variety of clinical conditions, includingneurodegenerative conditions involving synaptic loss, which conditionsmay include aging, Alzheimer's disease; amyotrophic lateral sclerosis;multiple sclerosis, glaucoma, myotonic dystrophy, Down syndrome;Parkinson's disease, Huntington's disease; and the like. The loss ofsynapses is inhibited by contacting neurons with agents that blockcomplement, including specific components, such as C1q.

In some aspects of the invention, methods are provided for screeningcandidate agents for the ability to modulate synaptic development,including synapse elimination. In one embodiment of the invention theneurons are neurons in the central nervous system. In anotherembodiment, the neurons are peripheral nervous system neurons. Screeningmethods are also provided for determining signaling molecules involvedin the synapse elimination pathway, e.g. molecules expressed byastrocytes, including immature astrocytes.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 demonstrates that astrocytes up-regulate C1q expression inneurons. A. Gene chip analysis (Affymetrix) of RNA prepared frompurified RGCs showed C1q was the only gene significantly (10-30 fold)up-regulated by astrocytes. B. RT-PCR validated that all three chains(A, B, C) of C1q were significantly up-regulated in RGCs upon exposureto astrocytes. C. C1q is highly expressed in retinal ganglion cells invivo. RT-PCR analysis of mRNA isolated from RGCs that were acutelyisolated from P5 retina (left lane), and perfused postnatal mouseretina.

FIG. 2 demonstrates that C1q is localized to developing CNS synapses invivo. A. Longitudinal cryosection of P5 mouse retina stained withanti-C1q. A punctate pattern of C1q immunoreactivity was observed in theganglion cell layer (GCL) and synaptic inner plexiform layer (IPL). B.Developmental localization of C1q to synapses in the IPL of the mouseretina. C. Double labeling of C1q (green) with pre synaptic marker SV2(red) demonstrate punctuate C1q-immunoreactivity in close proximity tosynaptic puncta in postnatal (P5), but not adult (P45) mouse cortex.Protein studies using more sensitive reagents (see, e.g., FIG. 33)demonstrates that C1q is localized to synaptic puncta in adult mousecortex as well. D. Higher magnification confocal image of C1q (green)and synaptic puncta in P5 mouse cortex.

FIG. 3 demonstrate that C1q-deficient mice have defects in synapticrefinement and eye-specific segregation. A. C1q KO mice at P10 (left tworows) and P30 (last two rows) have expanded ipsilateral projections(red) and significant intermingling (overlap) between RGC axons fromleft and right eyes (yellow) compared to littermate WT controls.Retinogeniculate projection patterns were visualized after injectingβcholera toxin conjugated to Alexa 594 (CTb-594) dye (red) and CTb-488(green) into left and right eyes of WT and C1q KO mice. Quantificationof the percentage of dLGN receiving overlapping inputs from theipsilateral eye in C1q KO vs WT controls at P10 (B) and P30 (C). C1q KOmice exhibit significantly more overlap than WT mice, regardless ofthreshold.

FIG. 4 demonstrates that LGN neurons remain multiply-innervated inC1q-deficient mice. A. Representative traces are of single LGN neuronsrecorded from a WT and C1q KO mouse. Superposition of the peaks of therapid inward current (AMPAR, −70 mV), and slower decaying outwardcurrent (NMDAR, +40 mV) represent the recruitment of individual axons.B. Recordings from LGN neurons of P30 mice in acute parasagittal brainslices. The optic tract was stimulated in small incremental intensitysteps, and measured the amplitude of evoked responses in LGN neurons(red) in the contralateral region adjacent to the optic tract in C1q KOsand age matched controls. CTβ labeled contralateral retinal projectionsare shown in green, and ipsilateral projections are in blue. C. 81% ofthe cells recorded were classified as unrefined (greater than 2 inputs)compared to 27% in age matched wild type controls (C1q KO n=21, WT n=30cells, p<0.001). D. Summary of response properties of control and C1q KOLGN neurons. C1q KOs remain multiply-innervated (average of 4±0.3inputs, n=21) compared to age-matched WT controls (average of 2.2±0.2inputs, n=30, p<0.001).

FIG. 5 illustrates that mice deficient in C3 also have defects insynapse elimination. A. Anterograde tracing experiments showing C3 KOmice at P30 exhibit significant overlap between RGC axons from left andright eyes (yellow) compared to littermate WT controls. B Quantificationof the percentage of dLGN receiving overlapping inputs from theipsilateral eye in C1q KO versus WT controls at P10 (B) and P30 (C). C1qKO mice exhibit significantly more overlap than WT mice, regardless ofthreshold. D. Electrophysiological recordings of P30-34 dLGN neuronsindicate that LGN neurons recorded from C3 KO mice remainmulti-innervated (non refined) and had similar response properties toC1q KO mice.

FIG. 6 illustrates that complement C3 is expressed at developing CNSsynapses. A Double immunohistochemistry with the synaptic antibody SV2,revealed that many C3-positive puncta co-localized with synaptic punctain the developing (P5), but not the adult brain (P60). B. Western blotanalysis of protein lysates prepared from perfused, developing cortex. Aclear band for iC3b (43 kD) was observed in postnatal cortex, and C3blevels were significantly down-regulated by P30.

FIG. 7 demonstrates that Complement—deficient mice have more synapses.A. Immunohistochemistry of pre-synaptic protein, vGLUT2 in the superiorcolliculi (SC) and dLGN of P16 C1q KO and control mice indicate anincrease in average intensity of staining in C1q KOs. Sections wereprocessed and immunostained in parallel and all images were collected atidentical cameral exposures. B. High resolution confocal microscopyimaging revealed a higher density of postsynaptic PSD95 puncta (green)in dLGNs of P16 C1q KO mice compared to littermate controls.

FIG. 8 demonstrates that C1q is localized to synapse in early stages ofglaucoma. Immohistochemistry of A. C1q and the postsynaptic protein, B.PSD95 in the RGC and C. synaptic layers of retinas from DBA/2J micevarious stages of disease (pre-glaucoma, early, moderate and latestage). Punctate C1q immunoreactivity (top row) in the IPL of retinasfrom DBA/2J mice with moderate (Grade III, 6-12 months) glaucoma andbefore significant RGC or synapse cell loss C1q staining is present inthe IPL, but less pronounced in retinas collected from retinas withsevere glaucoma and RGC death (as seen in DAPI staining of RGC cellbodies, bottom row).

FIG. 9 illustrates the localization of C1q within the synapseA.C1q-positive puncta in the IPL of postnatal mice were in closeapposition with synaptic puncta identified by co-immunostaining with preand post-synaptic markers, PSD95 (top) and SV2 (bottom). B. The synapticpattern of C1q immunoreactivity in the retina (left) was not detectedafter preadsorbing C1q antibodies with purified C1q protein (right).

FIG. 10 illustrates whole mount retinas stained with nuclear dye, DAPI,to demonstrate that there are no significant differences in the numberof cells in the peripheral or central regions of whole mount retinas inC1q KO and controls.

FIG. 11 illustrates by multielectrode recordings of RGC firing patternsthat C1q-deficient mice have normal retinal waves. In both WT and C1qKOretinas (P5), neighboring ganglion cells are more correlated in theirfiring than those located at further distances from one another.

FIG. 12 demonstrates that microglia, the resident phagocytes in thebrain, express receptors for complement (C3 and C1q). Phagocyticmicroglia in dLGN were stained with anti-CD68 at different developmentaltimepoints. CD68+ microglia most strongly label the dLGN at P15, a timeof active synapse elimination. B. An example of engulfment of RGCterminals (red, CTR) by CD68+ microglia (green) were observed in thedLGN during the synapse elimination period (P10-P15).

FIG. 13 illustrates the Complement pathway.

FIG. 14 demonstrates that synaptic C1q deposition followed by massivesynapse loss are the two earliest pathological features in the DBA2Jmouse glaucoma model

FIG. 15 demonstrates that C1q deficiency is strongly neuroprotective inglaucoma. 63% of wild-type mice but only 9% of C1q null mice havemoderate to severe (MOD-SEV) glaucoma.

FIG. 16 demonstrates that rabbit anti-Mouse C1q antibodies are specific.(A) Western blot of CNS tissue from wild type and C1q null animals withC1q polyclonal antibodies. (B) Immunostaining of CNS tissue with C1qmonoclonal antibodies.

FIG. 17 illustrates that C1q immunoreactivity increases in the agingmouse brain. 5× mosaic scan of C57BL/6 wild type mice.

FIG. 18 illustrates C1q protein expression in the aging mouse CNS. a, b,Time-course immunohistochemistry with slices from perfused brains fromthree different mouse strains confirmed that C1q protein levelsdramatically increase in an identical pattern with age throughout thebrain, independent of mouse strain and sex (a, female; b, male). c,Western blot analyses of mouse whole-brain homogenates (perfused mice)with two independent antibodies, either recognizing the C1q-A and -Bchains or the C1q-C chain, confirmed the dramatic age-dependent increaseof C1q protein in the mouse brain, when compared to the βTubulin loadingcontrols. Note that all available anti-C1q antibodies detect unspecificbands in Western blot experiments of mouse brain homogenates. The oneunspecific band directly above the C1q-specific band (arrow) varies invisibility between experiments. d, Western blot analysis of mousewhole-brain homogenates (perfused mice) confirmed that C1q proteinsimilarly increases with aging in the brains of male and female mice,when compared to the βTubulin loading controls. e, This sex-independentincrease of C1q protein (arrow head) with age was also confirmed byWestern blotting to be mouse-strain independent, when compared to theβTubulin loading controls (arrow: additional, unspecific band detectedby the antibody used). f, The increase with age in the brain isC1q-protein specific as other CNS proteins, like PSD-95, do not show asimilar regulation, when compared to the βTubulin loading controls. g,Like in the brain, C1q protein increases with age in spinal cordextracts from perfused mice, when compared to the βTubulin loadingcontrols. h, C1q-protein levels in the retina are moderate at youngpostnatal ages but, n strong contrast to brain and spinal cord, decreaseto below detection levels in adulthood and aging, when compared to theβTubulin loading controls. P—postnatal day; m—month; Scale bars: 2 mm.

FIG. 19 demonstrates that C3 protein is expressed in CNS tissuethroughout adulthood.

FIG. 20 demonstrates that C3 protein does not increase with aging in themurine brain. Western blot analysis of perfused, whole-brain homogenatesrevealed that, in contrast to C1q, C3 levels did not increase with agein the murine brain. Instead, C3 protein levels were high at earlypostnatal ages and decreased to constantly low levels at about one monthof age and after when compared to the βTubulin loading controls. Theseanti-C3 signals were specific as the corresponding band was not presentin brain homogenate from C3 KO mice (C3KO). j, Quantification of Westernblot-derived C3 signals, normalized to βTubulin loading controls,confirmed that C3 protein decreased from early postnatal ages to about 1month of age (C3 levels, normalized to P6 levels: P6: 1-fold, +/−0.14;P12: 0.43-fold+/−0.07; P20: 0.41-fold+/−0.05; P35: 0.12 fold+/−0.01; n=3animals/age. Significance: P12 versus P6: P=0.029; P20 versus P12:P=0.434; P35 versus P20: P=0.003, unpaired t-test). From about 1 monthof age and after, C3 protein levels were detectable at comparably lowlevels (P35 normalized to P6 levels: 0.12 fold+/−0.01; 3.5 month:0.26+/−0.07; 12 month: 0.17+/−0.01; 18 month: 0.23+/−0.02; 24 month:0.22+/−0.03; n=3 animals/age; 3.5 month versus P35: P=0.07; 12 monthversus 3.5 month: P=0.18; 18 month versus 12 month: P=0.50; 24 monthversus 18 month: P=0.88). P—postnatal day; m—month; per.—perfused;n.p.—not perfused; scale bars: a, b, 1 mm; c, d, 100 μm; ns—notsignificant, *P<0.05, **P<0.01. Values represent mean+/−S.E.M. Errorbars represent S.E.M.

FIG. 21 illustrates C1q immunoreactivity in the P6 mouse brain.

FIG. 22 provides a characterization of C1q-positive patches in the mousebrain. a, Representative immunohistochemistry image of a two year oldmouse brain slice (perfused animal), stained with the rabbit anti-mouseC1q monoclonal antibody. C1q-specific signals were detected in virtuallyall synaptic layers of the mouse brain, except the molecular layer ofthe Cerebellum (white arrow head). C1q-positive signals wereparticularly strong in the Hippocampus (blue arrow head), the PiriformCortex (yellow arrow head) and the Substantia Nigra (red arrow head).C1q-positive patches appeared throughout the brain (arrows). b, c,Higher magnification images depicting the variability in appearance ofthese C1q-positive patches (arrows). d, e, Confocal microscopy revealedthat the C1q-positive patches were neither particularly co-localizingwith microglia (d, Cx3CR1-GFP labeled microglia) nor astrocytes (e,ALdH1L1-GFP labeled astrocytes) but that the C1q-positive signals appeardiffuse in the neuropil around all cell types (large DAPI labeledneuronal cell bodies). f-k, Confocal microscopy co-localization analysesfor C1q and the excitatory synaptic protein VGluT1 (f-h) or theinhibitory synaptic protein VGAT (i-k) revealed that the overallsynaptic pattern was not altered in C1q-positive patches. WhereasC1q-immunoreactivity was increased in patch areas (f₁, g₁, i₁, j₁) overareas outside the patches (hi, k₁), both VGlut1 and VGATimmunoreactivities were unchanged inside or outside the C1q-positivepatches (inside patch area: g2, VGluT1 and j2, VGAT; outside patch area:h₂, VGluT1 and k₂, VGAT). Scale bars: a, 1 mm; b, 250 μm; c, 100 μm; d,e, f, l, 50 μm; g, h, j, k, 5 μm.

FIG. 23 demonstrates that C1q protein levels dramatically increase inthe aging mouse brain. A Western blot of brain homogenates is shown.

FIG. 24 demonstrates quantification of C1q protein levels dramaticallyincreasing in the aging mouse brain. (A) Quantification of Western blotsignals in FIG. 21. (B) Quantification of Western blot signals,normalized to β Tubulin loading controls. Quantification confirmed thatC1q protein dramatically increased with age to about 300 fold at 24month of age when normalized to postnatal day six (P6) levels(301+/−33.32, P=0.003, unpaired t-test; n=3 animals/age). Every ageanalyzed revealed a significant increase in C1q-protein levels whencompared to the next younger age from P6 to 18 month of age C1q levels,normalized to P6 levels: P6: 1-fold, +/−0.02; P12: 3-fold+/−0.24; P20:8-fold+/−0.60; P35: 12-fold+/−1.04; 3.5 month: 27-fold+/−3.73; 12 monthold: 59-fold+/−3.10; 18 month old: 204-fold+/−12.29; n=3 animals/age.Significance: P12 versus P6: P=0.019; P20 versus P12: P=0.0005; P35versus P20: P=0.019; 3.5 month versus P35: P=0.043; 12 month versus 3.5month: P=0.012; 18 month versus 12 month: P=0.0017). C1q levels in 24month old brains were not significantly different to the levels measuredat 18 month (P=0.104). P—postnatal day; m—month; per.—perfused; n.p.—notperfused; scale bars: a, b, 1 mm; c, d, 100 μm; ns—not significant,*P<0.05, **P<0.01. Values represent mean+/−S.E.M. Error bars representS.E.M.

FIG. 25 demonstrates that C1q protein levels do not increase in agingmouse serum.

FIG. 26 demonstrates that the increase in CNS C1q protein levels cannotbe explained by age-dependent leakage of the blood-brain barrier. perf,perfused. n.p., not perfused.

FIG. 27 demonstrates that C1q protein in the CNS is expressed bymicroglia at all developmental stages. a, b, Co-localizationimmunohistochemistry analysis confirmed that anti-C1q immunoreactivityin the perfused postnatal day six mouse brain was virtually exclusivelydetected in microglia, visualized by anti-GFP immunoreactivity in slicesfrom Cx3CR1-GFP+/− mice (a and b: costaining of C1q and Cx3CR1-GFP; a1and b1: C1q staining alone: a2 and b2: Cx3CR1-GFP staining alone; imagerepresentative for all brain regions; image location: midbrain; boxedarea in a, is shown as magnified image in b). c, d, Co-localizationimmunohistochemistry analysis revealed that anti-C1q immunoreactivity inthe perfused adult mouse brain was strongly detected in the neuropil andin all microglia (a and b: costaining of C1q and Cx3CR1-GFP; al and b1:C1q staining alone: a2 and b2: Cx3CR1-GFP staining alone; imagerepresentative for all brain regions; image location: midbrain; boxedarea in c, is shown as magnified image in d). Scale bars: a, 50 μm; b,d, 20 μm; c, 100 μm.

FIG. 28 demonstrates that C1q protein is detected in a subset of neuronsthroughout the murine brain. a, Immunohistochemistry analysis of a slicefrom an adult mouse brain (perfused, slightly modified processing:shortened fixation with 4% PFA and processed with a freezing, slidingmicrotome) enabled additional C1q-protein detection in a distinct subsetof neurons in certain areas of the brain. These C1q-positive neuronswere detected from about one month of age on and aging-independentthereafter. C1q-positive neurons were detected in the Hippocampus,Thalamus, Striatum and Midbrain, dependent of the location of the sliceswithin the brain. b, Detection of C1q-positive neurons in theHippocampus and Thalamus. c, Detection of C1q-positive neurons in theHippocampus, Thalamus and Midbrain. d, Detection of C1q-positive neuronsin the Hippocampus, Thalamus, Midbrain and Striatum. e, Detection ofC1q-positive neurons in the Thalamus and Striatum (mainly GlobusPallidus). Scale bars: a-e, 500 μm.

FIG. 29 demonstrates by C1q/parvalbumin co-staining that C1qimmunoreactive neurons are mainly inhibitory. a, hippocampus. b, globuspallidus.

FIG. 30 demonstrates that C1q protein is detected in a subset ofinhibitory neurons in distinct areas of the murine brain. a, b, c,Co-localization immunohistochemistry with anti-C1q- and the inhibitoryneuron marker anti-GABA antibodies confirmed that all C1q-positiveneurons are GABA-positive, identifying these as inhibitory neurons. AllC1q-positive neurons in the hippocampus (a), the SubstantiaNigra/Midbrain (b) and the Globus Pallidus/Striatum (c) are GABAergic(arrows). However, some GABAergic cells are not stained by theC1q-specific antibody (arrow heads). d, High-power, single-planeconfocal microscopy of the adult mouse Dentate Gyrus confirmedC1q-positive neurons as GABAergic. Anti-C1q immunoreactivityco-localized with anti-GABA immunoreactivity (arrow). Note the twoadditional GABAergic cells not stained by the anti-C1q antibody (arrowheads). a, b, c, and d: costaining of C1q (green) and GABA (red); a1,b1, c1, d1: C1q staining alone; a2, b2, c2, d2: GABA staining alone.Scale bars: a, 500 μm; b, c, 200 μm; d, 20 μm.

FIG. 31 illustrates that C1q protein dramatically increased with age inhippocampal synaptic layers in both mouse and human. a,Immunohistochemistry analysis confirmed comparably low levels ofanti-C1q-immunoreactivity (arrows), representing cellular signals, inthe hippocampus from postnatal day six mice. b, C1q-immunoreactivity wasdramatically increased in all synaptic layers of the 24 month old murinehippocampus, including the stratum radiatum (SR). However, signals wereparticularly strong in the Dentate Gyrus molecular layer (ML), thestratum lacunosum moleculare (SLM) and, to some degree, in the stratumoriens (SO). In addition, very strong anti-C1q immunoreactivity wasdetected in the neuropil surrounding the CA2 cell bodies (arrow head).c, d, representative anti-C1q immunoreactivity in human hippocampalslices from both infant (c) and aged (d) donors confirmed that C1qprotein also dramatically increased with age in synaptic layers of thehuman Hippocampus. As in mice, C1q protein was particularly enriched inthe stratum lacunosum moleculare (SLM) and the Dentate Gyrus molecularlayer (ML) but to a lesser extend in the stratum radiatum (SR). Inaddition to anti-C1q immunoreactivity in the neuropil, signals were alsodetected in blood vessels (arrows), indicating serum-derived C1q in thisnon-perfused tissue. Scale bars: a, b, c, d, 500 μm.

FIG. 32 demonstrates that C1q protein dramatically increased in theaging murine hippocampus. a, Time-course immunohistochemistry analysisof slices from perfused, early postnatal to old aged mouse brainsconfirmed that C1q protein levels dramatically increase with age insynaptic layers of the Hippocampus, including the stratum radiatum (SR).However, signals particularly increased in the Dentate Gyrus molecularlayer (ML), the stratum lacunosum moleculare (SLM) and in the stratumoriens (SO). In addition, very strong anti-C1q immunoreactivity wasdetected in the neuropil surrounding the CA2 cell bodies (arrow). b,Western blot analyses of mouse Hippocampus homogenates (perfused mice)confirmed the dramatic age-dependent increase of C1q protein (arrowhead) in the hippocampus, when compared to the βTubulin loading controls(arrow: additional, unspecific band detected by the antibody used). c,Like in whole brain homogenates, C3 protein levels (arrow head) in theHippocampus are comparably high at early postnatal stages but decreaseto constantly low levels from about 1 month of age onwards, whencompared to the βTubulin loading controls (arrow: additional, unspecificband detected by the antibody used). d, e, The increase with age in theHippocampus is C1q-protein specific as other CNS proteins, likesynaptotagmin (d) and GluR1 (e), do not show a similar regulation.P—postnatal day; m—month; Scale bars: 500 μm.

FIG. 33 demonstrates that C1q staining pattern is consistent throughoutentire adult hippocampus. sagittal sections. 1 is most medial, 5 is mostlateral.

FIG. 34 illustrates that C1q protein dramatically accumulates insynaptic layers of the aging human hippocampus. a-d, Anti-C1qimmunohistochemistry of human Hippocampus slices from infants (a, 7month old; c, 2 month old) and a 75 year old donor (b, d) confirmed thatC1q protein (a1, b1, c1, d1) dramatically increased with age in theneuropil of synaptic layers, in particular in the Dentate Gyrusmolecular layer (ML) and the stratum lacunosum moleculare (SLM), and toa lesser extend in the stratum radiatum (SR). Anti-Synaptophysin signals(a2, b2, c2, d2) were detected in all synaptic layers, independent ofage. DAPI (a3, b3, c3, d3) labeled the cell bodies. Arrows indicateserum-derived C1q-signals in blood vessels of this unperfused tissue. e,Western blot of human tissue samples confirmed the absolute specificityof the rabbit anti-human C1q antibody used for the human tissueimmunohistochemistry analysis. The antibody exclusively detected allsubunits of human C1q in both human serum (adult) and human hippocampalhomogenate from the 75 year old donor also analyzed byimmunohistochemistry in b, and d. No signals were detected in theC1q-depleted human serum sample, arguing for the absolute specificity ofthe antibody. a, b, c, and d: costaining of C1q (red), synaptophysin(green), and DAPI (blue); a1, b1, c1, d1: C1q staining alone; a2, b2,c2, d2: synaptophysin staining alone; a3, b3, c3, d3: DAPI stainingalone. kD-kilo Dalton; Scale bars: a, b, 500 μm; c, d, 100 μm.

FIG. 35 illustrates that C1q protein localizes to synapses in the adultand aged Hippocampus. a, b, Anti-C1q immunohistochemistry signalsvisualized by confocal microscopy are specific. Anti-C1qimmunoreactivity was only detected in the adult C1q wild-type DentateGyrus molecular layer (a) but not in the corresponding tissue from C1qKO littermate mice (b). c, d, Most anti-C1q signals were detected inclose proximity to synaptic protein signals in this high-power confocalmicroscopy co-localization study with the excitatory synaptic proteinVGluT1 (c) and the inhibitory synaptic protein Gephryn (d) (c:costaining of C1q (red) and VGluT1 (red); c1: Cq1 staining alone; c2:VGluT1 staining alone; d: costaining of C1q (red) and Gephryn (green);d1: C1q staining alone; d2: Gephryn staining alone). In addition,absolute co-localization of C1q with these synaptic proteins (arrows)was occasionally observed. These results suggested that C1q protein alsolocalizes to both excitatory and inhibitory synapses. e-h, Cryo-immunoelectron microscopy confirmed that C1q protein indeed localized to theoutside of both pre- and postsynaptic elements (arrows) in the DentateGyrus molecular layer of adult and aged mice. e, Anti-C1qimmunoreactivity, visualized with 12 nm gold particles, was detected inclose proximity to both pre-synaptic (green) and postsynaptic (brown)elements in tissue from adult mice. In addition, a few signals wereassociated with unidentified structures (arrow head). This single-planeimage analysis did not allow clear identification of all elementsvisible in each section. It is therefore impossible to classify anti-C1qimmunoreactivity associated with unidentified elements as synaptic orextra-synaptic (see panel f for explanation—anti-Synaptophysin signals).f, Dual-immuno-gold analysis in the Dentate Gyrus molecular layer ofaged mice. Anti-C1q immunoreactivity (12 nm gold particles, arrows) wasdetected in close proximity to both pre-synaptic (green) andpostsynaptic (brown) elements. In contrast, anti-Synaptophysinimmunoreactivity (6 nm gold particles, arrow heads) was detected insideclearly identifiable presynaptic terminals as well as associated withunidentifiable structures, likely reflecting presynaptic terminals. g,h, Anti-C1q cryo-immuno electron microscopy signals are specific.Anti-C1q immunoreactivity (12 nm gold particles) was only detected inthe adult C1q wild-type Dentate Gyrus molecular layer (g) but not in thecorresponding tissue from C1q KO littermate mice (h). In wild-typeanimals, C1q-immunoreactivity was detected in close proximity tosynaptic elements (arrows) as well as associated with unidentifiedstructures (arrow heads). Scale bars: a-d, 5 μm; e-h, 200 nm.

FIG. 36 illustrates that C1q-deficiency did not affect dendritic spinenumber or spine morphology in old aged mice. a, Representative image ofa Golgi-stained dendrite used for the dendritic spine quantificationanalysis. b-f, Tertiary Dentate Gyrus granule cell dendrites wereidentified and traced in Neurolucida (MBF Bioscience) and dendriticspines were manually identified, counted (n=4 animals/genotype, 9dendrites/animal) and quantified as number of spines per μm dendritelength. Spine type categorization was done according Peebles, C. et al.2010 PNAS 107(42):18173-78. No significant differences were detected foreither total spine number per μm dendrite length (b) or any of theindividual spine types counted (c-f). Error bars represent S.E.M.

FIG. 37 demonstrates that C1q-deficiency caused enhanced long-termpotentiation (LTP) in the Dentate Gyrus of adult mice. a, b, LTPanalysis in the Dentate Gyrus molecular layer of young postnatal (a,postnatal day 14 to 17) and adult (b, 3 month old) C1q KO (KO) versusC1q wild-type (WT) littermate mice. Adult C1q KO mice (b, circles)showed enhanced potentiation after tetanic stimulation when compared totheir wild-type (squares) littermates. Solid and hatched line indicatesinterval of significant difference; WT: 117.4+/−0.524 and KO:139.3+/−1.085 fEPSP (% baseline), P=0.044, 2-way repeated measures ANOVAwith Bonferoni posttest. In addition, adult C1q KO mice showed enhancedpost-tetanic potentiation when compared to their wild-type littermates(solid line, 1 minute after tetanus: WT: 149.1+/−7.2, KO: 167.1+/−6.7%of baseline). Potentiation in adult C1q KO mice also decayed faster thanin their wild-type littermates (P<0.001). This was in strong contrast toearly postnatal mice in which LTP levels, post-tetanic stimulation andLTP-decay were nearly identical between C1q KO and C1q wild-typelittermates (P=0.93). Each data point presented represents 1 minuteaverages of 4 individual fEPSPs taken at 15 second intervals. Postnatalday 14-17: n=6 (C1q WT), 6 (C1q KO); 3 month old: n=10 (C1q WT), 12 (C1qKO). Values represent mean+/−S.E.M.; Error bars represent S.E.M.

FIG. 38 demonstrates that C1q-deficient mice showed reduced cognitiveaging. a, b Morris Water Maze analysis, including reversal learning(days 8, 9, 10; escape latency shown). a, 3 month old adult C1q KO mice(KO), their wild-type littermates (WT), and strain matched control mice(bio controls, Bio Ctrl) performed all parts of the experiment withoutsignificant differences. b, Learning of the hidden platform location(days 1-7) was also mostly identical between 17 months old aged C1q KO,their wild-type littermates and young bio controls. Solely on day 7, thebio controls performed significantly better than the aged C1q wild-typemice (in seconds: Bio Ctrl: 90.29+/−8.996; WT; 145.8+/−15.19, P<0.01,repeated measure ANOVA and Bonferoni posthoc test). However, nosignificant difference in learning was detected between bio controls andaged C1q KO mice (in seconds: 126.2+/−13.48, P>0.05). On the first dayof the reversal learning task (day 8), the young bio controls learnedthe new location of the hidden platform faster than both aged C1q KO andwild-type littermate mice (in seconds: WT: 181.0+/−9.982; KO:175.2+/−9.095; Bio Ctrl: 129.6+/−13.94; WT versus Bio Ctrl: P<0.05; KOversus Bio Ctrl: P<0.05). However, aged C1q KO mice performed at the twosubsequent reversal learning days as fast as the young bio controls (day9, in seconds: KO: 120.8+/−11.69; Bio Ctrl: 115.8+/−13.11; P>0.05; andday 10, in seconds: KO: 115.2+/−11.49; Bio Ctrl: 102.7+/−15.7; P>0.05).This was in strong contrast to the aged C1q wild-type littermates whichshowed reduced reversal learning at both days, consistent with anage-related decline in cognitive flexibility (day 9, in seconds: WT:171.5+/−12.13; WT versus KO: P<0.05; WT versus Bio Ctrl: P<0.05; and day10, in seconds: WT: 153.0+/−12.91; WT versus KO: P>0.05; WT versus BioCtrl: P<0.05). There were no significant differences between all cohortsduring visible platform learning (day 11), indicating that all animalscan properly see. c, The C1q KO mice performed spontaneous alternationsin the Y-Maze task at all ages significantly above chance (50%), equallygood as the 3 month old bio controls. In contrast, 17 month oldwild-type littermate mice did not perform above chance levels,indicating age-related decrease in spatial working memory in the agedwild-type but not aged C1q KO littermates (at 17 month of age: WT:56.4%+/−3.91, P=0.116; KO: 64.60+/−2.83 S.E.M; P=0.0009; Bio Ctrl:61.72+/−3.39; P=0.0072, one-sample t-test). d, C1q KO mice showeddelayed age-related memory decline in the Novel Object Recognition task.Whereas all young adult (3 month old) mice performed this task withoutsignificant differences equally well, middle-aged (12 month old) C1qwild-type mice were no longer able to significantly differentiatebetween a novel and a familiar object, indicating an age-dependentdecline in recognition memory (in seconds: WT, familiar object:29.96+/−4.968 and WT, novel object: 45.14+/−5.818, P=0.062, pairedt-test). In strong contrast, middle aged C1q KO mice (12 month old) werestill able to learn the task, with similar performance as the young biocontrols (all in seconds: KO, familiar object: 36.33+/−7.497 and KO,novel object: 61.84+/−6.990, P=0.033; Bio Ctrl, familiar object:32.26+/−6.452 and Bio Ctrl, novel object: 52.47+/−8.070, P=0.049).However, at 17 month of age, both C1q wild-type and C1q KO mice failedto learn the task (all in seconds: WT, familiar object: 13.84+/−2.594and WT, novel object: 17.99+/−4.026, P=0.379; KO, familiar object:18.12+/−3.648 and KO, novel object: 19.75+/−2.947, P=0.646; Bio Ctrl,familiar object: 16.44+/−2.334 and Bio Ctrl, novel object:27.88+/−3.147, P=0.032). 3 month old: n=13 wild-type, 13 C1q KOlittermates; 10-12 month old: n=16 wild-type, 14 C1q KO littermates; 17month old: n=11 wild-type, 9 C1q KO littermates; bio controls: n=10;ns—not significant, *P<0.05, **P<0.01, *<0.001. Values representmean+/−S.E.M.; Error bars represent S.E.M.

FIG. 39 demonstrates that C1q-deficiency did not affect paired-pulsefacilitation in the murine hippocampus. a, b, Paired-pulse facilitationanalyses in the mouse Dentae Gyrus molecular layer from early postnatalmice (a, postnatal day 14-17) and adult mice (b, 3 month old) did notreveal any differences in presynaptic activity between C1q KO (KO) andtheir wild-type littermates (WT). al and b1: data for all inter-pulseinterval ranges recorded, a2 and b2: higher resolution of the shortinter-pulse interval data points. All data points represent groupaverages from C1q KO or C1q wild-type littermate cohorts. Postnatal day14-17: n=6 (C1q WT), 6 (C1q KO); 3 month old: n=10 (C1q WT), 12 (C1qKO); Error bars represent S.E.M.

FIG. 40 demonstrates that C1q-deficient mice were overall healthy andshowed normal behavior in basic behavioral tasks as well as in fearconditioning/fear extinction analysis. a, SHIRPA analysis confirmed thatbasic animal behavioral traits did not differ significantly betweenadult C1q KO mice (KO), C1q wild-type (WT) littermate mice and thestrain- and age-matched biological controls (Bio Ctrl). b, Open fieldanalysis confirmed that C1q KO mice have activity levels identical totheir wild-type littermates at both young adult and old ages. As to beexpected, the 3 month old biological control mice traveled slightlylonger distance than the 17 month old C1q KO and wild-type littermatemice (significant difference between C1qWT/KO and the bio controls at 5minutes: P<0.05, and 7 minutes: P<0.01; no significant difference at allother time points (P>0.05); Bonferoni posthoc test). c, d, ContextualFear conditioning (c) and Fear Extinction (d) analysis revealed nearlyidentical learning of C1q KO and C1q wild-type littermate mice at both 3month and 17 month of age. Both genotypes performed the FearConditioning task without significant statistical difference in freezing(c) and showed comparable ability to unlearn the conditioned response inthe Fear Extinction task (d). e-h, Supporting data for the Morris WaterMaze results presented in FIG. 4. e, g, During the probe trial, with theescape platform missing, both 3 month old (e) and 17 month old (h) C1qKO and C1q wild-type littermates as well as the young bio controlspreferred the target quadrant (TQ). f, h, For velocity, no significantgenotype difference was detectable (P>0.05), repeated measures ANOVA).

DETAILED DESCRIPTION OF THE INVENTION

Methods are provided for protecting or treating an individual sufferingfrom adverse effects of synapse loss, e.g. preventing additional synapseloss in an individual suffering from the synapse loss and the affectsthereof, prophylactically preventing synapse loss in an individual, e.g.an individual at risk for synapse loss, etc. It is shown herein thatimmature astrocytes in normal development produce a signal that inducesneurons to express specific complement proteins, thus enabling adevelopmental window during which synapse elimination occurs. Expressionof these proteins in development mirrors the period of developmentalsynaptogenesis, being off in embryonic brain and on at higher levels inpostnatal brain. Expression of these specific complement proteins isalso upregulated in the older adult brain and in neurodegenerativeconditions, thus enabling unwanted synapse elimination in theseaging-associated conditions.

These findings have broad implications for a variety of clinicalconditions, for example cognitive decline associated with aging wheresynapse loss is involved, e.g. natural aging and aging-associatedneurodegenerative conditions. Synapse loss is inhibited by contactingneurons with inhibitors or antagonists of the complement pathway. Forexample, inhibitors can block activation of the complement cascade, canblock the expression of specific complement proteins in neurons, caninterfere with signaling molecules that induce complement activation,can upregulate expression of complement inhibitors in neurons, andotherwise interfere with the role of complement in synapse loss. Theability to prevent synapse loss, e.g. in adult brains, has importantimplications for maintaining normal neuronal function in a variety ofneurodegenerative conditions.

Definitions

Synapse Loss.

Synapses are asymmetric communication junctions formed between twoneurons, or, at the neuromuscular junction (NMJ) between a neuron and amuscle cell. Chemical synapses enable cell-to-cell communication viasecretion of neurotransmitters, whereas in electrical synapses signalsare transmitted through gap junctions, specialized intercellularchannels that permit ionic current flow. In addition to ions, othermolecules that modulate synaptic function (such as ATP and secondmessenger molecules) can diffuse through gap junctional pores. At themature NMJ, pre- and postsynaptic membranes are separated by a synapticcleft containing extracellular proteins that form the basal lamina.Synaptic vesicles are clustered at the presynaptic release site,transmitter receptors are clustered in junctional folds at thepostsynaptic membrane, and glial processes surround the nerve terminal.

Synaptogenesis is a dynamic process. During development, more synapsesare established than ultimately will be retained. Therefore, theelimination of excess synaptic inputs is a critical step in synapticcircuit maturation. Synapse elimination is a competitive process thatinvolves interactions between pre- and postsynaptic partners. In theCNS, as with the NMJ, a developmental, activity-dependent remodeling ofsynaptic circuits takes place by a process that may involve theselective stabilization of coactive inputs and the elimination of inputswith uncorrelated activity. The anatomical refinement of synapticcircuits occurs at the level of individual axons and dendrites by adynamic process that involves rapid elimination of synapses. As axonsbranch and remodel, synapses form and dismantle with synapse eliminationoccurring rapidly.

In addition to the normal developmental loss, synapse loss is an earlypathological event common to many neurodegenerative conditions, and isthe best correlate to the neuronal and cognitive impairment associatedwith these conditions. Studies in the brains of patients withpre-clinical Alzheimer's disease (AD), as well as in transgenic animalmodels have shown that synaptic damage occurs early in diseaseprogression. This early disruption of synaptic connections in the brainresults in neuronal dysfunction that, in turn, leads to thecharacteristic symptoms of dementia and/or motor impairment observed inseveral neurodegenerative disorders.

Several molecules involved in AD and other neurodegenerative disordersplay an important role in synaptic function. For example, AβPP has apreferential localization at central and peripheral synaptic sites. Intransgenic mice, abnormal expression of mutant forms of AβPP results notonly in amyloid deposition, but also in widespread synaptic damage. Thissynaptic pathology occurs early and is associated with levels of solubleAβ1-42 rather than with plaque formation. Other neurodegenerativediseases where gene products have been shown to be closely associatedwith synaptic complexes include Huntington's disease (HD) and myotonicdystrophy (DM). Huntingtin is a membrane-bound protein with adistribution very similar to that of synaptic vesicle proteinsynaptophysin. Studies in human brain detected htt in perikarya of someneurons, neuropil, varicosities and as punctate staining likely to benerve endings. The serine/threonine kinase (DMK), which is the geneproduct of the DM gene, has been found to localize post-synaptically atthe neuromuscular junction of skeletal muscle and at intercalated discsof cardiac tissue. DMK was also found at synaptic sites in thecerebellum, hippocampus, midbrain and medulla. By “modulation” ofsynapse loss as used herein, it is meant that the number of synapseslost is either enhanced or suppressed as required in the specificsituation. As used herein, the term “modulator of synapse loss” refersto an agent that is able to alter synapse loss. Modulators include, butare not limited to, both “activators” and “inhibitors”. An “activator”or “agonist” is a substance that enhances synapse loss. Conversely, an“inhibitor” or “antagonist” decreases synapse loss. The reduction may becomplete or partial. As used herein, modulators include, withoutlimitation, C1q antagonists and agonists. Agonists and antagonists mayinclude proteins, nucleic acids, carbohydrates, antibodies, or any othermolecules that decrease the effect of a protein. The term “analog” isused herein to refer to a molecule that structurally resembles amolecule of interest but which has been modified in a targeted andcontrolled manner, by replacing a specific substituent of the referencemolecule with an alternate substituent. Compared to the startingmolecule, an analog may exhibit the same, similar, or improved utility.Synthesis and screening of analogs, to identify variants of knowncompounds having improved traits (such as higher potency at a specificreceptor type, or higher selectivity at a targeted receptor type andlower activity levels at other receptor types) is an approach that iswell known in pharmaceutical chemistry.

Complement.

Complement is a system of plasma proteins that interacts with the cellsurfaces of pathogens or cells to mark them for destruction byphagocytes. The complement system is made up of a large number ofdistinct plasma proteins, primarily produced by the liver. A number ofthese proteins are a class of proteases, called zymogens, which arethemselves activated by proteolytic cleavage. These zymogens can thus bewidely distributed without being active until activated by a localpathogen. The complement system thus is activated through a triggeredenzyme cascade.

The classical pathway is activated by the binding of the complementprotein C1q directly to the cell surface or to an antibody that is boundto the cell surface. C1q is a large multimeric protein of 460 kDaconsisting of 18 polypeptide chains (6 C1q A chains, 6 C1q B chains, and6 C1q C chains). C1r and C1s complement proteins to bind to the C1q tailregion to form the C1 complex. Binding of the C1q complex to the surfaceof a cell or to the complement binding domain of an antibody Fc regioninduces a conformational change in C1q that leads to activation of anautocatalytic enzymatic activity in C1r, which then cleaves C1s togenerate an active serine protease. Once activated, C1s cleaves C4, etc,leading to the complement cascade sequence. Ultimately this pathwayleads to the formation of a membrane attack complex which lyses andkills the affected cell. Normal cells, including neurons, expressmolecules such as CD59 that protect them from lysis or damage from themembrane attack complex and the C1 inhibitor (C1-INH) which dissociatesC1r and C1s from the active C1 complex.

Various complement proteins are expressed by neurons and glial cells invitro and in vivo. Their function in the brain is unknown. Theexpression of many of these complement proteins is upregulated by serumor inflammatory cytokines or after brain injury. Astrocytes in culturehave been reported to express C1q, C1r, C1s, C4, C2, and C3, as well asthe more terminal proteins. Neurons have been reported to express C4 andC3, but only to express C1q after brain injury.

Three pathways have been elucidated through which the complement cascadecan be initiated; classical, alternate and lectin Pathways. All threepathways merge through at common intersection, complement C3. C3 is anacute phase reactant. The liver is the main site of synthesis, althoughsmall amounts are also produced by activated monocytes and macrophages.A single chain precursor (pro-C3) of approximately 200 kD is foundintracellularly; the cDNA shows that it comprises 1,663 amino acids.This is processed by proteolytic cleavage into alpha and beta subunitswhich in the mature protein are linked by disulfide bonds. Pro-C3contains a signal peptide of 22 amino acid residues, the beta chain (645residues) and the alpha chain (992 residues). The 2 chains are joined by4 arginine residues that are not present in the mature protein. In thealternate pathway complement C3 undergoes spontaneous cleavage resultingin complement B binding to C3b. Diffusion of the Ba subunit results inan active alternate pathway C3 convertase (C3bBb). C3bBb is stabilizedby binding to properdin prior to merging.

Inhibition of Complement.

A number of molecules are known that inhibit the activity of complement.As described above, normal cells can produce proteins that blockcomplement activity, i.e., that naturally block complement activity,e.g. CD59, C1 inhibitor, etc. In some embodiments of the invention,complement is inhibited by upregulating expression of genes encodingsuch polypeptides. Modified molecules that block complement activation,e.g. dominant negative polypeptides of complement cascade proteins, arealso known, which can be exogenously provided to the cells as a solublepeptide or which can be ectopically expressed in cells by contacting thecells with a nucleic acid comprising the dominant negative polypeptide.Moreover, small molecule inhibitors of complement signaling have beenidentified which may be provided to the cells.

For example, the activity of the C1 complex (composed of 1 molecule ofC1q, 3 molecules of C1r, and 2 molecules of C1s) may be inhibited. Anyinhibitor of C1 complex function, e.g. inhibitors of binding of the C1complex to an antigen or pathogen, inhibitors of binding between C1q,C1r and C1s; inhibitors of C1r activation by C1q; inhibitors of C1scleavage by C1r; inhibitors C1s protease activity on C4, may be employedin the subject methods. For example, naturally occurring inhibitors ofthe C1 complex such as C1 Inhibitor and Chondroitin Sulfate Proteoglycan(CSPG) may be used. C1 Inhibitor is a member of the “serpin” family ofserine protease inhibitors; it is a heavily glycosylated plasma proteinthat prevents fluid-phase C1 activity by blocking the active site of C1rand C1s and dissociating them from C1q. CSPG binds to the complementprotein C1q and inhibits complex formation of C1 (Kirschfink et al.,1997, J Immunol 158 (3):1324-1331). Another example of inhibitors of C1complex are dominant negative polypeptide inhibitors, e.g. solubleversions of C1q receptors such as soluble CR1, soluble CR1 (sCR1),soluble cC1qR/caltreticulin, soluble gC1qR, soluble C1qRp, etc. Forexample, the mature protein of the most common allotype of CR1 contains1998 amino acid residues: an extracellular domain of 1930 residues, atransmembrane region of 25 residues, and a cytoplasmic domain of 43residues. The entire extracellular domain is composed of 30 repeatingunits (FIG. 2) referred to as short consensus repeats (SCRs) orcomplement control protein repeats (CCPRs), each consisting of 60 to 70amino acid residues. Recent data indicate that C1q binds specifically tohuman CR1. A soluble version of recombinant human CR1 (sCR1) lacking thetransmembrane and cytoplasmic domains has been produced and shown toretain all the known functions of the native CR1. Other C1q receptors(C1qR) have been described (see the review by McGreal and Gasque, 2002,Biochem Soc Trans 39 (Pt.6):1010-4), any of which may be modified toproduce dominant negative polypeptides that will bind C1q to inhibit C1complex binding to cells. Other examples of inhibitors of C1 complexthat find use in the subject methods include the synthetic peptide C1q Bchain helical region (Fryer et al., 1997); the sulfated polysaccharideFucan (Charreau et al., 1997); small molecule inhibitors of C1 complexactivity such as the C1 s inhibitors BCX-1470 (K-76 analog, Kaufman etal., 1995), K-76 derivatives (Sindelar et al. 1996; Tanaka 1996)C1s-INH-248 (Buerke et al. 2001. J immunol 167(9):5375-80), andthiopheneamidine-based inhibitors (Subasinghe et al. 2004. Biororg MedChem Lett. 14(12):3043-7); and glycosaminoglycans such as heparin (teVelthuis et al., 1996) and the highly sulfated low molecular weightheparin derivative LU 51198 (Gralinski et al., 1997).

As another example, the activity of the C5 convertase complex (C4b2a3b)may be inhibited. Any inhibitor of C5 convertase activity, e.g.inhibitors of binding of C4b, C2a, and C3b; cleavage of C5 into C5a andC5b by the C5 convertase, activity of C5a or C5b etc., may be used. Onesuch example of an inhibitor would be a dominant negative polypeptidebased on Decay accelerating factor (DAF). DAF (CD55) is composed of fourSCRs plus a serine/threonine-enriched domain that is capable ofextensive O-linked glycosylation. DAF is attached to cell membranes by aglycosyl phosphatidyl inositol (GPI) anchor and, through its ability tobind C4b and C3b, it acts by dissociating the C3 and C5 convertases.Soluble versions of DAF (sDAF) have been shown to inhibit complementactivation. Other examples inhibitors of C5 convertase activity includethe Streptomyces peptide Complestatin (Momota et al., 1991); syntheticpeptides such as C5aRAM (van Oostrum et al., 1996), C5a C-terminaloctapeptides (Kawai et al., 1992), C5a His67-modified C-terminaloctapeptide analogues (Or et al., 1992), and C089 (C5a hexapeptide,Konteatis et al., 1994); and small molecule antagonists such asL-156,602 (Tsuji et al., 1992) and FUT-175 (nafamstat mesilate, Inose rtal. 1997).

As another example, the activity of the C3 convertase complex (C3bBb3b)may be inhibited. Any inhibitor of C3 convertase activity, e.g.inhibitors of binding of C3b to Bb; cleavage of C3 into C3a and C3b bythe C3 convertase, activity of C3a or C3b etc., may be used. Examples ofsuch inhibitors include polypeptides such as cobra venom factor bCVFb(Jungi and McGregor, 1979), dominant negative inhibitors of DAF asdescribed above, synthetic peptide antagonists of C3a comprisingC-terminal modifications (Kretzschmar et al., 1992), and small moleculeantagonists such as FUT-175 (nafamstat mesilate, Inose et al. 1997).

Other examples of members of the complement cascade that may beinhibited include Factor B, which can be inhibited by, e.g., FactorB-related hexapeptides (Lesavre et al., 1982); Factor D, which can beinhibited by, e.g., DFP (Diisopropyl fluorophosphates, Cole et al.,1997); components C2, C3 and C4, which can be inhibited by, e.g., M5, afibrin(ogen)olytic proteinase from Crotalus molossus molossus (Chen andRael, 1997)

In addition to known compounds, suitable inhibitors can be screened bymethods described herein. Any convenient agent that modulates theactivity of any member or complex so as to inhibit the activity of thecomplement pathway may be employed.

Conditions of Interest

By “neurological” or “cognitive” function as used herein, it is meantthat the decrease of synapses in the brain enhances the patient'sability to think, function, etc. As used herein, the term “subject”encompasses mammals and non-mammals. Examples of mammals include, butare not limited to, any member of the mammalian class: humans, non-humanprimates such as chimpanzees, and other apes and monkey species; farmanimals such as cattle, horses, sheep, goats, swine; domestic animalssuch as rabbits, dogs, and cats; laboratory animals including rodents,such as rats, mice and guinea pigs, and the like. The term does notdenote a particular age or gender.

Among the conditions of interest for the present methods of inhibitingsynapse loss are included a variety of aging-associatedneurodegenerative conditions, e.g. naturally occurring aging,Alzheimer's disease, Down syndrome, Huntington's disease, amyotrophiclateral sclerosis, multiple sclerosis, myotonic dystrophy, glaucoma,Parkinson's disease; and the like. Such conditions benefit fromadministration of inhibitors of complement, including inhibitors of C1q,which allow maintenance, or reduced loss, of synapses. In someinstances, where there has been neuronal loss, it may be desirable toenhance neurogenesis as well, e.g. through administration of agents orregimens that increase neurogenesis, transplantation of neuronalprogenitors, etc. Agents that enhance synaptogenesis, e.g. such asthrombospondins, may also be administered.

Aging is the accumulation of changes in an organism over time. In thenervous system, it is accompanied by stereotypical structural andneurophysiological changes and variable degrees of cognitive decline.Included in these changes are synapse loss and the loss of neuronalfunction that results. Synapse loss begins at at least about age 20, andmay or may not be accompanied by cognitive decline. Typically, ifcognitive decline occurs, it is a modest disruption of memory oftenreferred to as “age-associated cognitive impairment” or “mild cognitiveimpairment” (MCI) that manifests as problems with memory or other mentalfunctions such as planning, following instructions, or making decisionsthat have worsened over time while overall mental function and dailyactivities are not impaired. Thus, although significant neuronal deathdoes not typically occur, neurons in the aging brain are vulnerable tosub-lethal age-related alterations in structure, synaptic integrity, andmolecular processing at the synapse, all of which impair cognitivefunction.

Moreover, the exponential buildup of C1q on aging, senescent synapsesmakes these synapses highly vulnerable to the action of the complementcascade. As a result, almost any injury of the aging brain (sepsis,trauma, toxin, ischemia, etc.) may cause activation of the complementcascade at C1q-coated aging synapses, leading to neurodegeneration and,in some instances, triggering other neurodegenerative diseases such asthose described below, thus exacerbating cognitive/memory lossassociated with normal aging. As such, any individual at the age inwhich synapses begin to be lost, e.g. over the age 20, may benefit fromthe administration of complement inhibitors.

Alzheimer's disease is a progressive, inexorable loss of cognitivefunction associated with an excessive number of senile plaques in thecerebral cortex and subcortical gray matter, which also containsβ-amyloid and neurofibrillary tangles consisting of tau protein. Thecommon form affects persons >60 yr old, and its incidence increases asage advances. It accounts for more than 65% of the dementias in theelderly.

The cause of Alzheimer's disease is not known. The disease runs infamilies in about 15 to 20% of cases. The remaining, so-called sporadiccases have some genetic determinants. The disease has an autosomaldominant genetic pattern in most early-onset and some late-onset casesbut a variable late-life penetrance. Environmental factors are the focusof active investigation.

In the course of the disease, synapses, and ultimately neurons are lostwithin the cerebral cortex, hippocampus, and subcortical structures(including selective cell loss in the nucleus basalis of Meynert), locuscaeruleus, and nucleus raphae dorsalis. Cerebral glucose use andperfusion is reduced in some areas of the brain (parietal lobe andtemporal cortices in early-stage disease, prefrontal cortex inlate-stage disease). Neuritic or senile plaques (composed of neurites,astrocytes, and glial cells around an amyloid core) and neurofibrillarytangles (composed of paired helical filaments) play a role in thepathogenesis of Alzheimer's disease. Senile plaques and neurofibrillarytangles occur with normal aging, but they are much more prevalent inpersons with Alzheimer's disease.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, invariablyfatal neurological disease that attacks motor neurons. Muscular weaknessand atrophy and signs of anterior horn cell dysfunction are initiallynoted most often in the hands and less often in the feet. The site ofonset is random, and progression is asymmetric. Cramps are common andmay precede weakness. Rarely, a patient survives 30 yr; 50% die within 3yr of onset, 20% live 5 yr, and 10% live 10 yr. Diagnostic featuresinclude onset during middle or late adult life and progressive,generalized motor involvement without sensory abnormalities. Nerveconduction velocities are normal until late in the disease.

A decrease in cell body area, number of synapses and total synapticlength has been reported in even normal-appearing neurons of the ALSpatients. It has been suggested that when the plasticity of the activezone reaches its limit, a continuing loss of synapses can lead tofunctional impairment. Preventing synapse loss may maintain neuronfunction in these patients.

Down Syndrome is a chromosomal disorder usually resulting in mentalretardation, a characteristic facies, and many other typical features,including microcephaly and short stature. In about 95% of cases, thereis an extra whole chromosome 21. At autopsy, adult Down syndrome brainsshow the typical microscopic findings of Alzheimer's disease, and manypersons also develop the associated clinical signs.

Parkinson's Disease is an idiopathic, slowly progressive, degenerativeCNS disorder characterized by slow and decreased movement, muscularrigidity, resting tremor, and postural instability. In primaryParkinson's disease, the pigmented neurons of the substantia nigra,locus caeruleus, and other brain stem dopaminergic cell groups are lost.The cause is not known. The loss of substantia nigra neurons, whichproject to the caudate nucleus and putamen, results in depletion of theneurotransmitter dopamine in these areas. Onset is generally after age40, with increasing incidence in older age groups.

Secondary parkinsonism results from loss of or interference with theaction of dopamine in the basal ganglia due to other idiopathicdegenerative diseases, drugs, or exogenous toxins. The most common causeof secondary parkinsonism is ingestion of antipsychotic drugs orreserpine, which produce parkinsonism by blocking dopamine receptors.Less common causes include carbon monoxide or manganese poisoning,hydrocephalus, structural lesions (tumors, infarcts affecting themidbrain or basal ganglia), subdural hematoma, and degenerativedisorders, including striatonigral degeneration and

Myotonic dystrophy is an autosomal dominant multisystem disordercharacterized by dystrophic muscle weakness and myotonia. The moleculardefect is an expanded trinucleotide (CTG) repeat in the 3′ untranslatedregion of the myotonin-protein kinase gene on chromosome 19q. Symptomscan occur at any age, and the range of clinical severity is broad.Myotonia is prominent in the hand muscles, and ptosis is common even inmild cases. In severe cases, marked peripheral muscular weakness occurs,often with cataracts, premature balding, hatchet facies, cardiacarrhythmias, testicular atrophy, and endocrine abnormalities (eg,diabetes mellitus). Mental retardation is common. Severely affectedpersons die by their early 50s.

Glaucoma is a common neurodegenerative disease that affects retinalganglion cells (RGCs). Substantial effort is being expended to determinehow RGCs die in glaucoma. Evidence supports the existence ofcompartmentalised degeneration programs in synapses and dendrites,including RGCs. Recent data, from in vitro studies and from an inheritedmouse model of glaucoma, suggest that molecularly distinct degenerativepathways underlie the destruction of RGC somata and RGC axons. Invarious neurodegenerative diseases, axons, dendrites and synapses oftendegenerate well before the cells die, and there is increasing evidencethat this is important for the production of clinical symptoms andsigns.

Huntington's disease (HD) is a hereditary progressive neurodegenerativedisorder characterized by the development of emotional, behavioral, andpsychiatric abnormalities; loss of previously acquired intellectual orcognitive functioning; and movement abnormalities (motor disturbances).The classic signs of HD include the development of chorea—orinvoluntary, rapid, irregular, jerky movements that may affect the face,arms, legs, or trunk—as well as the gradual loss of thought processingand acquired intellectual abilities (dementia). There may be impairmentof memory, abstract thinking, and judgment; improper perceptions oftime, place, or identity (disorientation); increased agitation; andpersonality changes (personality disintegration). Although symptomstypically become evident during the fourth or fifth decades of life, theage at onset is variable and ranges from early childhood to lateadulthood (e.g., 70s or 80s).

HD is transmitted within families as an autosomal dominant trait. Thedisorder occurs as the result of abnormally long sequences or “repeats”of coded instructions within a gene on chromosome 4 (4p16.3). Theprogressive loss of nervous system function associated with HD resultsfrom loss of neurons in certain areas of the brain, including the basalganglia and cerebral cortex.

Multiple Sclerosis is characterized by various symptoms and signs of CNSdysfunction, with remissions and recurring exacerbations. The mostcommon presenting symptoms are paresthesias in one or more extremities,in the trunk, or on one side of the face; weakness or clumsiness of aleg or hand; or visual disturbances, e.g. partial blindness and pain inone eye (retrobulbar optic neuritis), dimness of vision, or scotomas.Other common early symptoms are ocular palsy resulting in double vision(diplopia), transient weakness of one or more extremities, slightstiffness or unusual fatigability of a limb, minor gait disturbances,difficulty with bladder control, vertigo, and mild emotionaldisturbances; all indicate scattered CNS involvement and often occurmonths or years before the disease is recognized. Excess heat mayaccentuate symptoms and signs.

The course is highly varied, unpredictable, and, in most patients,remittent. At first, months or years of remission may separate episodes,especially when the disease begins with retrobulbar optic neuritis.However, some patients have frequent attacks and are rapidlyincapacitated; for a few the course can be rapidly progressive.

The methods of the invention can find use in combination with cell ortissue transplantation to the central nervous system, where such graftsinclude neural progenitors such as those found in fetal tissues, neuralstem cells, embryonic stem cells or other cells and tissues contemplatedfor neural repair or augmentation. Neural stem/progenitor cells havebeen described in the art, and their use in a variety of therapeuticprotocols has been widely discussed. For example, inter alia, U.S. Pat.No. 6,638,501, Bjornson et al.; U.S. Pat. No. 6,541,255, Snyder et al.;U.S. Pat. No. 6,498,018, Carpenter; U.S. Patent Application 20020012903,Goldman et al.; Palmer et al. (2001) Nature 411(6833):42-3; Palmer etal. (1997) Mol Cell Neurosci. 8(6):389-404; Svendsen et al. (1997) Exp.Neurol. 148(1):135-46 and Shihabuddin (1999) Mol Med Today.5(11):474-80; each herein specifically incorporated by reference.

Neural stem and progenitor cells can participate in aspects of normaldevelopment, including migration along well-established migratorypathways to disseminated CNS regions, differentiation into multipledevelopmentally- and regionally-appropriate cell types in response tomicroenvironmental cues, and non-disruptive, non-tumorigenicinterspersion with host progenitors and their progeny. Human NSCs arecapable of expressing foreign transgenes in vivo in these disseminatedlocations. A such, these cells find use in the treatment of a variety ofconditions, including traumatic injury to the spinal cord, brain, andperipheral nervous system; treatment of degenerative disorders includingAlzheimer's disease, Huntington's disease, Parkinson's disease;affective disorders including major depression; stroke; and the like. Bysynapse loss enhancers, the functional connections of the neurons areenhances, providing for an improved clinical outcome.

Methods of Treatment

The methods of the invention provide for modulating synapse loss throughadministering agents that are agonists or antagonists of complement.Without being bound by theory, the data provided herein demonstrate thatimmature astrocytes induce expression of C1q proteins in neurons duringdevelopment. During the developmental process of synapse elimination,this C1q expression can be coupled with a signal for complementactivation, e.g. β-amyloid, APP, cytokines such as IFNγ, TNFα, and thelike, thereby eliminating specific synapses. This development pathwaymay be inappropriately activated in neurodegenerative disease, resultingin the undesirable loss of synapses. By administering agents that altercomplement activation, synapses can be maintained that would otherwisebe lost. Such agents include C1q inhibitors, agents that upregulateexpression of native complement inhibitors, agents that down-regulateC1q synthesis in neurons, agents that block complement activation,agents that block the signal for complement activation, and the like.

The methods promote improved maintenance of neuronal function inconditions associated with synapse loss. The maintenance of neuralconnections provides for functional stabilization and functionalimprovement in neurodegenerative disease relative to untreated patients.The prevention of synapse loss may comprise at least a measurableimprovement relative to a control lacking such treatment, for example atleast a 10% improvement in the number of synapses, at least a 20%improvement, at least a 50% improvement, or more.

Methods of the invention reduce the loss of synapses, e.g. the rate ofloss, or the total number of synapses lost, where a decrease wouldotherwise occur. In some instances, inhibiting synapse loss with agentsof the invention maintains the number of synapses that were present atthe time of first treatment. In other words, inhibiting synapse lossstabilizes the numbers of synapse such that no additional synapses arelost.

In some instances, synapse loss is associated with a decline in neuronalfunction. As such, inhibiting synapse loss with agents of the inventionreduces the decline in neuronal function, i.e. the rate or extent, by,e.g., 30% or more, e.g. 40%, 50%, 60% or more, such as 70%, 80%, or 90%or more, for example, 95% or 100% or more, i.e. such that the loss ofneuronal function is negligible, In other words, inhibiting synapse lossmay be said to maintain neuronal function, e.g. at the level of functionat the time of first treatment with an inhibitor of synapse loss orbetter, i.e. to improve neuronal function. Neuronal function in humanscan be measured by any convenient method, e.g. nerve stimulation toelectrically excite peripheral nerves; somatosensory evoked potentialtechniques to measure processing of sensory input by the brain;electromyography to measure electrical activity in muscles; andtranscranial magnetic stimulation, a technique used to investigate theoutput of the motor cortex to the muscles.

In some instances, synapse loss and the subsequent decline in neuronalfunction is associated with cognitive decline. By “cognition” it ismeant the ability of a person to think, e.g. pay attention, remember,produce and understand language, solve problems, and make decisions. By“cognitive decline”, it is meant a decline in the ability of theindividual to perform these mental tasks. Inhibiting synapse loss withagents of the invention reduces the rate of cognitive decline by, e.g.,30% or more, e.g. 40%, 50%, 60% or more, such as 70%, 80%, or 90% ormore, for example, 95% or 100% or more, i.e. such that cognitive declineis negligible. In other words, inhibiting synapse loss may be said toprevent a cognitive decline and maintain cognition, e.g. at the level ofcognition at the time of first treatment with an inhibitor of synapseloss. Cognitive decline can be measured by any convenient method, e.g.tests of memory or other mental functions such as planning, followinginstructions, or decision-making.

The agents of the present invention are administered at a dosage thatdecreases synapse loss while minimizing any side-effects. It iscontemplated that compositions will be obtained and used under theguidance of a physician for in vivo use. The dosage of the therapeuticformulation will vary widely, depending upon the nature of the disease,the frequency of administration, the manner of administration, theclearance of the agent from the host, and the like.

The effective amount of a therapeutic composition to be given to aparticular patient will depend on a variety of factors, several of whichwill be different from patient to patient. Utilizing ordinary skill, thecompetent clinician will be able to optimize the dosage of a particulartherapeutic or imaging composition in the course of routine clinicaltrials.

Therapeutic agents, e.g. inhibitors of complement, activators of geneexpression, etc. can be incorporated into a variety of formulations fortherapeutic administration by combination with appropriatepharmaceutically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants, gels, microspheres, and aerosols.As such, administration of the compounds can be achieved in variousways, including oral, buccal, rectal, parenteral, intraperitoneal,intradermal, transdermal, intrathecal, nasal, intracheal, etc.,administration. The active agent may be systemic after administration ormay be localized by the use of regional administration, intramuraladministration, or use of an implant that acts to retain the active doseat the site of implantation.

One strategy for drug delivery through the blood brain barrier (BBB)entails disruption of the BBB, either by osmotic means such as mannitolor leukotrienes, or biochemically by the use of vasoactive substancessuch as bradykinin. The potential for using BBB opening to targetspecific agents is also an option. A BBB disrupting agent can beco-administered with the therapeutic compositions of the invention whenthe compositions are administered by intravascular injection. Otherstrategies to go through the BBB may entail the use of endogenoustransport systems, including carrier-mediated transporters such asglucose and amino acid carriers, receptor-mediated transcytosis forinsulin or transferrin, and active efflux transporters such asp-glycoprotein. Active transport moieties may also be conjugated to thetherapeutic or imaging compounds for use in the invention to facilitatetransport across the epithelial wall of the blood vessel. Alternatively,drug delivery behind the BBB is by intrathecal delivery of therapeuticsor imaging agents directly to the cranium, as through an Ommayareservoir.

Pharmaceutical compositions can include, depending on the formulationdesired, pharmaceutically-acceptable, non-toxic carriers of diluents,which are defined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, buffered water, physiologicalsaline, PBS, Ringer's solution, dextrose solution, and Hank's solution.In addition, the pharmaceutical composition or formulation can includeother carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenicstabilizers, excipients and the like. The compositions can also includeadditional substances to approximate physiological conditions, such aspH adjusting and buffering agents, toxicity adjusting agents, wettingagents and detergents.

The composition can also include any of a variety of stabilizing agents,such as an antioxidant for example. When the pharmaceutical compositionincludes a polypeptide, the polypeptide can be complexed with variouswell-known compounds that enhance the in vivo stability of thepolypeptide, or otherwise enhance its pharmacological properties (e.g.,increase the half-life of the polypeptide, reduce its toxicity, enhancesolubility or uptake). Examples of such modifications or complexingagents include sulfate, gluconate, citrate and phosphate. Thepolypeptides of a composition can also be complexed with molecules thatenhance their in vivo attributes. Such molecules include, for example,carbohydrates, polyamines, amino acids, other peptides, ions (e.g.,sodium, potassium, calcium, magnesium, manganese), and lipids.

Further guidance regarding formulations that are suitable for varioustypes of administration can be found in Remington's PharmaceuticalSciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985).For a brief review of methods for drug delivery, see, Langer, Science249:1527-1533 (1990).

The pharmaceutical compositions can be administered for prophylacticand/or therapeutic treatments. Toxicity and therapeutic efficacy of theactive ingredient can be determined according to standard pharmaceuticalprocedures in cell cultures and/or experimental animals, including, forexample, determining the LD50 (the dose lethal to 50% of the population)and the ED50 (the dose therapeutically effective in 50% of thepopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit large therapeutic indices are preferred.

The data obtained from cell culture and/or animal studies can be used informulating a range of dosages for humans. The dosage of the activeingredient typically lines within a range of circulating concentrationsthat include the ED50 with low toxicity. The dosage can vary within thisrange depending upon the dosage form employed and the route ofadministration utilized.

The pharmaceutical compositions described herein can be administered ina variety of different ways. Examples include administering acomposition containing a pharmaceutically acceptable carrier via oral,intranasal, rectal, topical, intraperitoneal, intravenous,intramuscular, subcutaneous, subdermal, transdermal, intrathecal, andintracranial methods.

For oral administration, the active ingredient can be administered insolid dosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. The activecomponent(s) can be encapsulated in gelatin capsules together withinactive ingredients and powdered carriers, such as glucose, lactose,sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonate.Examples of additional inactive ingredients that may be added to providedesirable color, taste, stability, buffering capacity, dispersion orother known desirable features are red iron oxide, silica gel, sodiumlauryl sulfate, titanium dioxide, and edible white ink. Similar diluentscan be used to make compressed tablets. Both tablets and capsules can bemanufactured as sustained release products to provide for continuousrelease of medication over a period of hours. Compressed tablets can besugar coated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric-coated for selectivedisintegration in the gastrointestinal tract. Liquid dosage forms fororal administration can contain coloring and flavoring to increasepatient acceptance.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.

The components used to formulate the pharmaceutical compositions arepreferably of high purity and are substantially free of potentiallyharmful contaminants (e.g., at least National Food (NF) grade, generallyat least analytical grade, and more typically at least pharmaceuticalgrade). Moreover, compositions intended for in vivo use are usuallysterile. To the extent that a given compound must be synthesized priorto use, the resulting product is typically substantially free of anypotentially toxic agents, particularly any endotoxins, which may bepresent during the synthesis or purification process. Compositions forparental administration are also sterile, substantially isotonic andmade under GMP conditions.

The compositions of the invention may be administered using anymedically appropriate procedure, e.g. intravascular (intravenous,intraarterial, intracapillary) administration, injection into thecerebrospinal fluid, intracavity or direct injection in the brain.Intrathecal administration maybe carried out through the use of anOmmaya reservoir, in accordance with known techniques. (F. Balis et al.,Am J. Pediatr. Hematol. Oncol. 11, 74, 76 (1989).

Where the therapeutic agents are locally administered in the brain, onemethod for administration of the therapeutic compositions of theinvention is by deposition into or near the site by any suitabletechnique, such as by direct injection (aided by stereotaxic positioningof an injection syringe, if necessary) or by placing the tip of anOmmaya reservoir into a cavity, or cyst, for administration.Alternatively, a convection-enhanced delivery catheter may be implanteddirectly into the site, into a natural or surgically created cyst, orinto the normal brain mass. Such convection-enhanced pharmaceuticalcomposition delivery devices greatly improve the diffusion of thecomposition throughout the brain mass. The implanted catheters of thesedelivery devices utilize high-flow microinfusion (with flow rates in therange of about 0.5 to 15.0 μl/minute), rather than diffusive flow, todeliver the therapeutic composition to the brain and/or tumor mass. Suchdevices are described in U.S. Pat. No. 5,720,720, incorporated fullyherein by reference.

The effective amount of a therapeutic composition to be given to aparticular patient will depend on a variety of factors, several of whichwill be different from patient to patient. A competent clinician will beable to determine an effective amount of a therapeutic agent toadminister to a patient. Dosage of the agent will depend on thetreatment, route of administration, the nature of the therapeutics,sensitivity of the patient to the therapeutics, etc. Utilizing LD50animal data, and other information, a clinician can determine themaximum safe dose for an individual, depending on the route ofadministration. Utilizing ordinary skill, the competent clinician willbe able to optimize the dosage of a particular therapeutic compositionin the course of routine clinical trials. The compositions can beadministered to the subject in a series of more than one administration.For therapeutic compositions, regular periodic administration willsometimes be required, or may be desirable. Therapeutic regimens willvary with the agent, e.g. some agents may be taken for extended periodsof time on a daily or semi-daily basis, while more selective agents maybe administered for more defined time courses, e.g. one, two three ormore days, one or more weeks, one or more months, etc., taken daily,semi-daily, semi-weekly, weekly, etc.

Formulations may be optimized for retention and stabilization in thebrain. When the agent is administered into the cranial compartment, itis desirable for the agent to be retained in the compartment, and not todiffuse or otherwise cross the blood brain barrier. Stabilizationtechniques include cross-linking, multimerizing, or linking to groupssuch as polyethylene glycol, polyacrylamide, neutral protein carriers,etc. in order to achieve an increase in molecular weight.

Other strategies for increasing retention include the entrapment of theagent in a biodegradable or bioerodible implant. The rate of release ofthe therapeutically active agent is controlled by the rate of transportthrough the polymeric matrix, and the biodegradation of the implant. Thetransport of drug through the polymer barrier will also be affected bycompound solubility, polymer hydrophilicity, extent of polymercross-linking, expansion of the polymer upon water absorption so as tomake the polymer barrier more permeable to the drug, geometry of theimplant, and the like. The implants are of dimensions commensurate withthe size and shape of the region selected as the site of implantation.Implants may be particles, sheets, patches, plaques, fibers,microcapsules and the like and may be of any size or shape compatiblewith the selected site of insertion.

The implants may be monolithic, i.e. having the active agenthomogenously distributed through the polymeric matrix, or encapsulated,where a reservoir of active agent is encapsulated by the polymericmatrix. The selection of the polymeric composition to be employed willvary with the site of administration, the desired period of treatment,patient tolerance, the nature of the disease to be treated and the like.Characteristics of the polymers will include biodegradability at thesite of implantation, compatibility with the agent of interest, ease ofencapsulation, a half-life in the physiological environment.

Biodegradable polymeric compositions which may be employed may beorganic esters or ethers, which when degraded result in physiologicallyacceptable degradation products, including the monomers. Anhydrides,amides, orthoesters or the like, by themselves or in combination withother monomers, may find use. The polymers will be condensationpolymers. The polymers may be cross-linked or non-cross-linked. Ofparticular interest are polymers of hydroxyaliphatic carboxylic acids,either homo- or copolymers, and polysaccharides. Included among thepolyesters of interest are polymers of D-lactic acid, L-lactic acid,racemic lactic acid, glycolic acid, polycaprolactone, and combinationsthereof. By employing the L-lactate or D-lactate, a slowly biodegradingpolymer is achieved, while degradation is substantially enhanced withthe racemate. Copolymers of glycolic and lactic acid are of particularinterest, where the rate of biodegradation is controlled by the ratio ofglycolic to lactic acid. The most rapidly degraded copolymer has roughlyequal amounts of glycolic and lactic acid, where either homopolymer ismore resistant to degradation. The ratio of glycolic acid to lactic acidwill also affect the brittleness of in the implant, where a moreflexible implant is desirable for larger geometries. Among thepolysaccharides of interest are calcium alginate, and functionalizedcelluloses, particularly carboxymethylcellulose esters characterized bybeing water insoluble, a molecular weight of about 5 kD to 500 kD, etc.Biodegradable hydrogels may also be employed in the implants of thesubject invention. Hydrogels are typically a copolymer material,characterized by the ability to imbibe a liquid. Exemplary biodegradablehydrogels which may be employed are described in Heller in: Hydrogels inMedicine and Pharmacy, N. A. Peppes ed., Vol. III, CRC Press, BocaRaton, Fla., 1987, pp 137-149.

In some embodiments, methods of the invention further comprise the stepof administering to the patient a therapy for a neurodegenerativecondition, i.e. the neurodegenerative condition associated with thesynapse loss. In such embodiments, any convenient therapy may beprovided. For example, in methods in which the patient has sufferedsynapse loss as a result of Alzheimer's Disease, the methods may furthercomprise administering a therapy for Alzheimer's Disease. Any convenientAlzheimer's Disease therapy may be provided, for example, memantine, acholinesterase inhibitor such as donepezil, rivastigmine, or galantamin,anti-depressants, anti-anxiety medications to reduce agitation oraggression, antipsychotic medications to reduce suspicion or paranoia,etc,

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

Gene Delivery

One approach for modulating synapse loss involves gene therapy. In suchmethods, anti-sense or RNAi sequences encoding C1q or fragments thereofare introduced into the neurons, and expressed as a means of decreasingC1q expression in the targeted synapses. To genetically modify neuronsthat are protected by the BBB, two general categories of approaches havebeen used. In one type of approach, cells are genetically altered,outside the body, and then transplanted somewhere in the CNS, usually inan area inside the BBB. In the other type of approach, genetic “vectors”are injected directly into one or more regions in the CNS, togenetically alter cells that are normally protected by the BBB. Itshould be noted that the terms “transfect” and “transform” are usedinterchangeably herein. Both terms refer to a process which introduces aforeign gene (also called an “exogenous” gene) into one or morepreexisting cells, in a manner which causes the foreign gene(s) to beexpressed to form corresponding polypeptides.

A preferred approach introduces into the CNS a source of a desirablesequence, by genetically engineering cells within the CNS. This has beenachieved by directly injecting a genetic vector into the CNS, tointroduce foreign genes into CNS neurons “in situ” (i.e., neurons whichremain in their normal position, inside a patient's brain or spinalcord, throughout the entire genetic transfection or transformationprocedure).

Useful vectors include viral vectors, which make use of the lipidenvelope or surface shell (also known as the capsid) of a virus. Thesevectors emulate and use a virus's natural ability to (i) bind to one ormore particular surface proteins on certain types of cells, and then(ii) inject the virus's DNA or RNA into the cell. In this manner, viralvectors can deliver and transport a genetically engineered strand of DNAor RNA through the outer membranes of target cells, and into the cellscytoplasm. Gene transfers into CNS neurons have been reported using suchvectors derived from herpes simplex viruses (e.g., European Patent453242, Breakfield et al 1996), adenoviruses (La Salle et al 1993), andadeno-associated viruses (Kaplitt et al 1997).

Non-viral vectors typically contain the transcriptional regulatoryelements necessary for expression of the desired gene, and may includean origin of replication, selectable markers and the like, as known inthe art. The non-viral genetic vector is then created by adding, to agene expression construct, selected agents that can aid entry of thegene construct into target cells. Several commonly-used agents includecationic lipids, positively charged molecules such as polylysine orpolyethylenimine, and/or ligands that bind to receptors expressed on thesurface of the target cell. For the purpose of this discussion, theDNA-adenovirus conjugates described by Curiel (1997) are regarded asnon-viral vectors, because the adenovirus capsid protein is added to thegene expression construct to aid the efficient entry of the geneexpression construct into the target cell.

In cationic gene vectors, DNA strands are negatively charged, and cellsurfaces are also negatively charged. Therefore, a positively-chargedagent can help draw them together, and facilitate the entry of the DNAinto a target cell. Examples of positively-charged transfection agentsinclude polylysine, polyethylenimine (PEI), and various cationic lipids.The basic procedures for preparing genetic vectors using cationic agentsare similar. A solution of the cationic agent (polylysine, PEI, or acationic lipid preparation) is added to an aqueous solution containingDNA (negatively charged) in an appropriate ratio. The positive andnegatively charged components will attract each other, associate,condense, and form molecular complexes. If prepared in the appropriateratio, the resulting complexes will have some positive charge, whichwill aid attachment and entry into the negatively charged surface of thetarget cell. The use of liposomes to deliver foreign genes into sensoryneurons is described in various articles such as Sahenk et al 1993. Theuse of PEI, polylysine, and other cationic agents is described inarticles such as Li et al 2000 and Nabel et al 1997.

An alternative strategy for introducing DNA into target cells is toassociate the DNA with a molecule that normally enters the cell. Thisapproach was demonstrated in liver cells in U.S. Pat. No. 5,166,320 (Wuet al 1992). An advantage of this approach is that DNA delivery can betargeted to a particular type of cell, by associating the DNA with amolecule that is selectively taken up by that type of target cell. Alimited number of molecules are known to undergo receptor mediatedendocytosis in neurons. Known agents that bind to neuronal receptors andtrigger endocytosis, causing them to enter the neurons, include (i) thenon-toxic fragment C of tetanus toxin (e.g., Knight et al 1999); (ii)various lectins derived from plants, such as barley lectin (Horowitz etal 1999) and wheat germ agglutinin lectin (Yoshihara et al 1999); and,(iii) certain neurotrophic factors (e.g., Barde et al 1991). At leastsome of these endocytotic agents undergo “retrograde” axonal transportwithin neuron. The term “retrograde”, in this context, means that thesemolecules are actively transported, by cellular processes, from theextremities (or “terminals”) of a neuron, along an axon or dendrite,toward and into the main body of the cell, where the nucleus is located.This direction of movement is called “retrograde”, because it runs inthe opposite direction of the normal outward (“anterograde”) movement ofmost metabolites inside the cell (including proteins synthesized in thecell body, neurotransmitters synthesized by those proteins, etc.).

Compound Screening

In some aspects of the invention, candidate agents are screened for theability to modulate synapse loss ability and to inhibit cognitivedecline, which agents may include candidate complement inhibitors,variants, fragments, mimetics, agonists and antagonists. Such compoundscreening may be performed using an in vitro model, a geneticallyaltered cell or animal, or purified protein. A wide variety of assaysmay be used for this purpose. In one embodiment, compounds that arepredicted to be antagonists or agonists of complement, includingspecific complement proteins, e.g. C1q, and complement activatingsignals, e.g. β-amyloid, APP, etc. are tested in an in vitro culturesystem, as described below.

For example, candidate agents may be identified by known pharmacology,by structure analysis, by rational drug design using computer basedmodeling, by binding assays, by cell-free assays and the like for theirability to inhibit complement activity, where the ability of an agent toinhibit complement signaling is indicative of the ability of that agentto inhibit synapse loss and cognitive decline. Various in vitro modelsmay be used to determine whether a compound binds to, or otherwiseaffects complement activity. Such candidate compounds may used tocontact neurons in an environment permissive for synapse loss. Suchcompounds may be further tested in any convenient in vitro or in vivomodel for an effect on synapse loss, and/or for an effect on cognitivedecline, e.g. as described in the examples section below.

Screening may also be performed for molecules produced by astrocytes,e.g. immature astrocytes, which induce C1q expression in neurons. Insuch assays, co-cultures of neurons and astrocytes are assessed for theproduction or expression of molecules that induce C1q expression. Forexample, blocking antibodies may be added to the culture to determinethe effect on induction of C1q expression in neurons.

Synapse loss is quantitated by administering the candidate agent toneurons in culture, and determining the presence of synapses in theabsence or presence of the agent. In one embodiment of the invention,the neurons are a primary culture, e.g. of RGCs. Purified populations ofRGCs are obtained by conventional methods, such as sequentialimmunopanning. The cells are cultured in suitable medium, which willusually comprise appropriate growth factors, e.g. CNTF; BDNF; etc. Theneural cells, e.g. RCGs, are cultured for a period of time sufficientallow robust process outgrowth and then cultured with a candidate agentfor a period of about 1 day to 1 week. In many embodiments, the neuronsare cultured on a live astrocyte cell feeder in order to inducesignaling for synapse loss. Methods of culturing astrocyte feeder layersare known in the art. For example, cortical glia can be plated in amedium that does not allow neurons to survive, with removal ofnon-adherent cells.

For synapse quantification, cultures are fixed, blocked and washed, thenstained with antibodies specific synaptic proteins, e.g. synaptotagmin,etc. and visualized with an appropriate reagent, as known in the art.Analysis of the staining may be performed microscopically. In oneembodiment, digital images of the fluorescence emission are with acamera and image capture software, adjusted to remove unused portions ofthe pixel value range and the used pixel values adjusted to utilize theentire pixel value range. Corresponding channel images may be merged tocreate a color (RGB) image containing the two single-channel images asindividual color channels. Co-localized puncta can be identified using arolling ball background subtraction algorithm to remove low-frequencybackground from each image channel. Number, mean area, mean minimum andmaximum pixel intensities, and mean pixel intensities for allsynaptotagmin, PSD-95, and colocalized puncta in the image are recordedand saved to disk for analysis.

The term “agent” as used herein describes any molecule, e.g. protein orpharmaceutical, with the capability of modulating synapse loss,particularly through the complement pathway. Candidate agents alsoinclude genetic elements, e.g. anti-sense and RNAi molecules to inhibitC1q expression, and constructs encoding complement inhibitors, e.g. CD59, and the like. Candidate agents encompass numerous chemical classes,though typically they are organic molecules, including small organiccompounds having a molecular weight of more than 50 and less than about2,500 daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof. Generally a plurality of assay mixtures are run inparallel with different agent concentrations to obtain a differentialresponse to the various concentrations. Typically one of theseconcentrations serves as a negative control, i.e. at zero concentrationor below the level of detection.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs. Test agents can be obtained from libraries, such asnatural product libraries or combinatorial libraries, for example.

Libraries of candidate compounds can also be prepared by rationaldesign. (See generally, Cho et al., Pac. Symp. Biocompat. 305-16, 1998);Sun et al., J. Comput. Aided Mol. Des. 12:597-604, 1998); eachincorporated herein by reference in their entirety). For example,libraries of phosphatase inhibitors can be prepared by syntheses ofcombinatorial chemical libraries (see generally DeWitt et al., Proc.Nat. Acad. Sci. USA 90:6909-13, 1993; International Patent PublicationWO 94/08051; Baum, Chem. & Eng. News, 72:20-25, 1994; Burbaum et al.,Proc. Nat. Acad. Sci. USA 92:6027-31, 1995; Baldwin et al., J. Am. Chem.Soc. 117:5588-89, 1995; Nestler et al., J. Org. Chem. 59:4723-24, 1994;Borehardt et al., J. Am. Chem. Soc. 116:373-74, 1994; Ohlmeyer et al.,Proc. Nat. Acad. Sci. USA 90:10922-26, all of which are incorporated byreference herein in their entirety.)

A “combinatorial library” is a collection of compounds in which thecompounds comprising the collection are composed of one or more types ofsubunits. Methods of making combinatorial libraries are known in theart, and include the following: U.S. Pat. Nos. 5,958,792; 5,807,683;6,004,617; 6,077,954; which are incorporated by reference herein. Thesubunits can be selected from natural or unnatural moieties. Thecompounds of the combinatorial library differ in one or more ways withrespect to the number, order, type or types of modifications made to oneor more of the subunits comprising the compounds. Alternatively, acombinatorial library may refer to a collection of “core molecules”which vary as to the number, type or position of R groups they containand/or the identity of molecules composing the core molecule. Thecollection of compounds is generated in a systematic way. Any method ofsystematically generating a collection of compounds differing from eachother in one or more of the ways set forth above is a combinatoriallibrary.

A combinatorial library can be synthesized on a solid support from oneor more solid phase-bound resin starting materials. The library cancontain five (5) or more, preferably ten (10) or more, organic moleculesthat are different from each other. Each of the different molecules ispresent in a detectable amount. The actual amounts of each differentmolecule needed so that its presence can be determined can vary due tothe actual procedures used and can change as the technologies forisolation, detection and analysis advance. When the molecules arepresent in substantially equal molar amounts, an amount of 100 picomolesor more can be detected. Preferred libraries comprise substantiallyequal molar amounts of each desired reaction product and do not includerelatively large or small amounts of any given molecules so that thepresence of such molecules dominates or is completely suppressed in anyassay.

Combinatorial libraries are generally prepared by derivatizing astarting compound onto a solid-phase support (such as a bead). Ingeneral, the solid support has a commercially available resin attached,such as a Rink or Merrifield Resin. After attachment of the startingcompound, substituents are attached to the starting compound.Substituents are added to the starting compound, and can be varied byproviding a mixture of reactants comprising the substituents. Examplesof suitable substituents include, but are not limited to, hydrocarbonsubstituents, e.g. aliphatic, alicyclic substituents, aromatic,aliphatic and alicyclic-substituted aromatic nuclei, and the like, aswell as cyclic substituents; substituted hydrocarbon substituents, thatis, those substituents containing nonhydrocarbon radicals which do notalter the predominantly hydrocarbon substituent (e.g., halo (especiallychloro and fluoro), alkoxy, mercapto, alkylmercapto, nitro, nitroso,sulfoxy, and the like); and hetero substituents, that is, substituentswhich, while having predominantly hydrocarbyl character, contain otherthan carbon atoms. Suitable heteroatoms include, for example, sulfur,oxygen, nitrogen, and such substituents as pyridyl, furanyl, thiophenyl,imidazolyl, and the like. Heteroatoms, and typically no more than one,can be present for each carbon atom in the hydrocarbon-basedsubstituents. Alternatively, there can be no such radicals orheteroatoms in the hydrocarbon-based substituent and, therefore, thesubstituent can be purely hydrocarbon.

Compounds that are initially identified by any screening methods can befurther tested to validate the apparent activity. The basic format ofsuch methods involves administering a lead compound identified during aninitial screen to an animal that serves as a model for humans and thendetermining the effects on synapse loss. The animal models utilized invalidation studies generally are mammals. Specific examples of suitableanimals include, but are not limited to, primates, mice, and rats.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

Example 1

Methods

RGC Gene Expression Analysis Using Affymetrix GeneChip Arrays:

Purified RGCs were plated at a density of ˜100,000 cells per well in6-well dishes and cultured in the presence or absence of a liveastrocyte feeding layer for four days. Total RNA was harvested usingRNeasy Mini Kit (Qiagen). cDNA was synthesized from 2 μg total RNA usingthe Gibco BRL Superscript Choice system and a T7-(dT)24 primer[5′-GGCCAG-TGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3′ (SEQ ID NO:1)].Biotinylated cRNA target was prepared by T7 linear amplification usingthe Bioarray RNA Transcripts Labeling Kit (Enzo) followed byfragmentation. Target was hybridized to Affymetrix Test-2 GeneChiparrays to assess target performance and the Rat U34 genome GeneChiparray set following standard Affymetrix protocols. Microarray data wasgenerated in paired triplicates from three independent cultured RGCpreparations. Assessment of the quality of sample data and changes ingene expression were analyzed with MicroArray Suite 5.0 software. Genesidentified as changing in expression in response to astrocytes increasedor decreased at least 2-fold in all three replicates.

Semiquantitative RT-PCR:

Total RNA was prepared as above from RGCs cultured alone or with anastrocyte feeding layer, astrocyte conditioned medium or 5 μg/ml TSP1for six days. 1 μg total RNA was reverse-transcribed using RETROScript(Ambion) and 1/20th of resulting cDNA product was used for PCR. Thefollowing PCR primers were used: F-rC1qB(5′-cggaattcccttctctgccctgaggacgg-3′ (SEQ ID NO:2)), R-rC1qB(5′-cgggat-cctttctgcatgcggtctcggtc-3′ (SEQ ID NO:3)), F-mC1qA(5′-cggaattcgacaaggtcc-tcaccaaccag-3′ (SEQ ID NO:4)), R-mC1qA(5′-cgggatccggggtccttctcgatcc-3′ (SEQ ID NO:5), F-mC1qC(5′-ccgggggagccaggtgtggag-3′ (SEQ ID NO:6)), R-mC1qC(5′-gcacaggttggc-cgtatgcg-3′ (SEQ ID NO:7)). GAPDH primers were added toall reactions as an internal control (gapdh-s:5′-GGTCTTACTCCTTGGAGGCCATGT-3′ (SEQ ID NO:8); gapdh-as:5′-GACCCCTTCA-T-TGACCTCAACTACA-3′ (SEQ ID NO:9)). 2×PCR Mater Mix kit(Promega) was used as supplied by manufacturer with the exception of theaddition of 4 mM MgCl2 (5.5 mM final) to the C1qC reaction mix. PCRprogram was as follows: initial denaturation for 4 min at 94° C.; cycledenaturation for 1 min at 94° C., annealing for 30s at 55° C., andextension for 30 sec at 72° C. (30 cycles total); final extension for 10min at 72° C. The PCR products were fractionated on 1.5% agarose gel andvisualized by ethidium bromide staining. Results shown arerepresentative of three biological replicates.

Preparation of Astrocytes.

Cortical glia were prepared as described (Ullian et al., 2001). Briefly,P1-P2 cortices were papain-digested and plated in tissue culture flasks(Falcon) in a medium that does not allow neurons to survive (Dulbecco'sModified Eagle Medium, fetal bovine serum (10%), penicillin (100 U/ml),streptomycin (100 μg/ml), glutamine (2 mM) and Na-pyruvate (1 mM). After4 days non-adherent cells were shaken off of the monolayer and cellswere incubated another 2-4 days to allow monolayer to refill.

Medium was replaced with fresh medium containing AraC (10 μM) andincubated for 48 hours. Astrocytes were trypsinized and plated onto24-well inserts (Falcon, 1.0 μm) or 10 cm tissue culture dishes.

Purification and Culture of RGCs.

RGCs were purified by sequential immunopanning to greater than 99.5%purity from P5 mice or Sprague-Dawley rats (Simonsen Labs, Gilroy,Calif.), as previously described (Barres et al., 1988). Approximately30,000 RGCs were cultured per well in 24-well plates (Falcon) on glass(Assistant) or Aclar 22C (Allied Signal) coverslips coated withpoly-D-lysine (10 μg/ml) followed by laminin (2 μg/ml). RGCs werecultured in 600 μl of serum-free medium, modified from Bottenstein andSato (1979), containing Neurobasal (Gibco), bovine serum albumin,selenium, putrescine, triiodothyronine, transferrin, progesterone,pyruvate (1 mM), glutamine (2 mM), CNTF (10 ng/ml), BDNF (50 ng/ml),insulin (5 μg/ml), and forskolin (10 μM). Recombinant human BDNF andCNTF were generously provided by Regeneron Pharmaceuticals. TTX andPicrotoxin from RBI. All other reagents were obtained from Sigma.

Labeling of Retinogeniculate Afferents.

Mouse pups were anesthetized with inhalant isofluorane. Mice receivedintravitreal injections of Cholera Toxin-subunit (CTβ) conjugated toAlexa 488 (green label) in the left eye, and CTβ conjugated to Alexa 594(red label) into the right eye (2-3 μl; 0.5% in sterile saline;Molecular Probes, Eugene Oreg.; CT). 24 hours later mice weretranscardially perfused with 4% paraformaldyhyde (ages in textcorrespond to age at sacrifice), brain tissue was postfixed overnight,cryoprotected in 30% sucrose and then sectioned coronally at 40 μm,mounted onto gelatin-coated slides and coverslipped with Vectashield(Vector Laboratories; Burlingame, Calif.).

Image Quantification and Preparation of Photomicrographs.

Images were digitally acquired with a 3900×3600 pixel color CCD camera(Axiocam; Zeiss; Thornwood, N.Y.). Universal gains and exposures wereestablished for each label. Raw images of the dLGN were imported toPhotoshop (Adobe) and cropped to exclude the vLGN and IGL, then thedegree of left and right eye axon overlap was quantified using themulti-threshold protocol described in Torborg et al. (2005). Thistechnique is designed to compare overlap across a range of signal:noisevalues in WT versus transgenic mice. This approach best allows fordirect statistical comparison of overlap between various strains of miceat different ages and has been used by others as well (Pak et al.,2004). After quantification, images were imported to Photoshop (Adobe)for adjustments to intensity, cropping, and alignment. In some cases,artifact was removed from outside the boundaries of the dLGN.

The Classical Complement Cascade Mediates Developmental CNS SynapseElimination.

During development, activity-dependent competition between axons forsynaptic territory causes permanent removal of synaptic connections, butwhat determines which synapses will be eliminated? Excess numbers ofsynapses are first generated to establish the initial wiring pattern ofthe brain, but the formation of mature, precise neural circuits requiresthe selective elimination and pruning of inappropriate synapticconnections.

Much of our understanding of developmental synapse elimination in thebrain come from studies carried out in the developing visual system. Theretinogeniculate system has proven an excellent model system forstudying CNS developmental synapse elimination in vivo. In all binocularanimals, axons from retinal ganglion cells (RGCs) terminate in distinctnon-overlapping eye-specific domains in the dorsal lateral geniculatenucleus (dLGN). This segregation of retinogeniculate projections intoeye-specific territories occurs over a specific and well characterizedperiod in postnatal development, and reflects the elimination ofthousands of synapses within incorrect LGN territory and the expansionof axon terminals and formation of synapses within the correct eyespecific regions. Synapse elimination also occurs within monocularregions of the LGN during a two week period spanning eye opening (P8-P30in mouse). Initially, neurons in the rodent dLGN are innervated bymultiple (>10) RGC axons, but by the third week of postnataldevelopment, each dLGN neurons receive stable inputs from 1-2 RGC axons.Much like the neuromuscular junction in the peripheral nervous system,this developmental shift in synaptic convergence represents thepermanent elimination of inappropriate retinogeniculate synapses, andthe maintenance and strengthening of appropriate synaptic connections.

The appearance of astrocytes at synapses in the postnatal braincoincides with this dynamic period of synapse formation and elimination.It has been estimated that fine processes of astrocytes can ensheath asmany as 40,000 synapses, and recent research indicate a pivotal role forastrocyte-derived signals in the development of structural andfunctional of synapses in the brain. Thrombospondin was recentlyidentified as a critical astrocyte-secreted synaptogenic signal, andastrocytes likely secrete other signals that control the development andstabilization of functional synapses.

Here we identify an unexpected and novel role for astrocytes and theclassical complement cascade in CNS synapse elimination. The complementsystem is part of the innate immune system, and is our first line ofdefense against pathogens and infection. Gene chip studies revealed thatonly one gene was profoundly up-regulated in neurons by astrocytes;complement protein C1q. C1q's best known role in the immune system is topromote elimination of dead cells, pathogens, and debris. The classicalcascade is a triggered enzyme cascade activated by the binding(opsonization) of C1q directly to the membrane surface. C1q opsonizationcan eliminate unwanted cells by “tagging” them for removal by residentphagocytes, or by further activating the downstream proteases, whichultimately leads to the cleavage of the major complement protein, C3.Like C1q, activated C3 fragments (C3b, C3ib) can directly opsonize cellsfor phagocytosis, or eliminate unwanted cells via terminal activation ofthe cascade and the formation of the lytic membrane attack complex.

We hypothesized that complement opsonizes or “tags” synapses forselective elimination during development. The findings reported hereinidentify C1q and the classical complement cascade as a novel andimportant mediator of synapse elimination in the developing visualsystem. In the adult CNS, synapse loss often occurs long before clinicalsymptoms in many neurodegenerative diseases. We also provide evidencethat this complement-dependent mechanism of synapse elimination may berecapitulated in early stages of neurodegenerative diseases, such asglaucoma.

Results

Astrocytes Up-Regulate C1q in CNS Neurons.

We used a genomic approach to screen candidate neuronal genes that areregulated by astrocytes. We took advantage of our ability to growpurified retinal ganglion cells (RGCs) in the complete absence of gliaor other retinal cell types. RGCs cultured for several days below afeeding layer of astrocytes have 7-fold more functional synapses thanRGCs cultured alone. We used this established experimental paradigm toinvestigate astrocyte-dependent neuronal gene expression. RNA wascollected from postnatal RGCs that had been grown for one week in thepresence or absence of astrocytes, and target RNA was hybridized to anAffymetrix gene chip as previously described.

Surprisingly, only one neuronal gene was significantly up-regulated byastrocytes. This gene was identified as complement C1q, an immune systemprotein thought not to be normally expressed in the brain. C1q is alarge multimeric secreted protein composed of six identical subunitswith globular heads and long collagen-like tails (Kishore and Reid,2000). C1q consists of 18 polypeptide chains (6 C1q A chains, 6 C1q Bchains, and 6 C1q C chains). We found that all 3 subunits of C1q wereup-regulated in purified RGCs by 10-30-fold upon exposure to astrocytefeeding layer (FIG. 1A). The astrocyte-induced increase in C1q A, B, andC mRNA levels in RGC neurons was verified using semi-quantitative RT-PCR(FIG. 1B). Consistent with our in vitro experiments, C1q is highlyexpressed in retinal ganglion cells in vivo. RT-PCR analysis of mRNAisolated from RGCs that were acutely isolated (P5) indicate expressionof C1q (FIG. 1C). In addition, C1q mRNA was detected in samples ofperfused postnatal mouse retina. C1q levels were highest in the earlypostnatal retina (P5-P10), and declined precipitously after P15 (FIG.1C). This developmental expression pattern for astrocyte-induced C1qgene expression in neurons corresponds to the appearance of astrocytesin the postnatal CNS, and the period of synaptic refinement of retinalaxons in the visual system, and suggests the possibility that C1q has afunctional role in synaptic pruning.

C1q is Expressed at Synapses in the Developing CNS.

We performed immunostaining experiments in cryosections of rodent tissueat different developmental timepoints. Using several different C1qantisera, we found a bright, punctate pattern C1q immunoreactivitythroughout the developing brain and retina. Punctate C1qimmunoreactivity was highly localized to the synaptic inner plexiformlayer (IPL) of postnatnal mouse retinas, and was also observed in asubset of developing RGCs (FIG. 2A). Consistent with the expressionpattern of C1q mRNA in the postnatal retina (FIG. 1C), we found that C1qprotein expression and synaptic localization follow a similardevelopmental pattern (FIG. 2B). In addition, many C1q-positive punctain the IPL were in close apposition with synaptic puncta identified byco-immunostaining with pre and post-synaptic markers (FIG. 9A).

C1q protein is also highly expressed in synaptic regions of earlypostnatal rodent brain (P4-P10) (FIG. 2C). Double labeling with synapticantibodies such as SV2, demonstrate punctate C1q-immunoreactivity inclose proximity to synaptic puncta (FIG. 2D). As in the retina, thesynaptic pattern of C1q immunoreactivity in the brain was not observedin adult rodent brain sections, suggesting that C1q is localized tosynapses in developing, but not adult brain. This synaptic pattern ofC1q immunoreactivity was not detected when the same antibodies were usedto stain brain sections prepared from C1q A-deficient mice, or afterpreadsorbing C1q antibodies with purified C1q protein (FIG. 9B).

C1q is Required for Normal Retinogeniculate Refinement.

To determine whether C1q play a functional role in developmental synapseelimination in vivo, we focused on the development of theretino-geniculate synapse. Using a combination of neuroanatomical andelectrophysiological techniques, we investigated synaptic refinement andsynapse elimination in the dLGN of mice that lack the A chain of C1q(C1q KO). These mice have no gross neuroanatomical or behavioraldefects, despite their inability to express functional C1q protein, orto activate the classical complement cascade.

To visualize the pattern of retinogeniculate projections, we performedanterograde tracing of RGC afferents by injecting the β subunit ofcholera toxin conjugated to Alexa 594 (CTβ-594) dye (red) and CTR-Alexa488 (green) into respective left and right eyes of wild type and C1q KOmice at several postnatal ages (P5, P10 and P30). In the mouse LGN,eye-specific territories are established between P4-P8, such that byP10, axons from the ipsilateral eye have segregated into a smalleye-specific patch in the medial dLGN. LGN inputs are further refinedover the next two postnatal weeks, such that by P30, left and right eyesare completely segregated in to tight eye-specific territories, withminimal intermingling between RGC axons originating from left and righteyes.

We found that C1qA KO mice have significant defects in LGN refinementand segregation. At P10, the amount of dLGN territory occupied bycontralateral retinal projections was notably larger, and in many cases,the ipsilateral projections appeared more diffuse in C1q KOs compared towild type mice and litter mate controls (FIG. 3A, column a vs b).Consistent with these observations, the dLGN in C1q KO mice havesignificant defects in eye-specific segregation. We observed significantoverlap between left and right eye RGC projections in the dLGN of C1qKOs (FIG. 3A, column a vs b, bottom row). These data were quantifiedusing a well-established threshold-independent method of analysis, andindicate that C1q KOs had a significantly higher percentage ofoverlapping projections at all thresholds examined (Insert updated statsn=) (FIG. 3B).

Surprisingly, significant segregation defects were still evident injuvenile C1q KO mice (FIG. 3B, column c vs d, bottom row). Normally, byP30, there is very little overlap between the two eyes (<3% of dLGN haveoverlapping projections), but as shown in FIG. 10, C1q KO mice hadsignificant overlapping retinal projections, and these effects werethreshold-independent. The phenotype at P30 was similar to that observedat P10, suggesting that many LGN neurons remain binocularly innervatedlong after the bulk of eye-specific segregation has occurred in themouse (by P10). The pattern of RGC inputs to the dLGN appeared normal inC1q KO mice at P4-P5, a time point before significant segregationoccurs, which suggests that refinement defects in C1q KO mice are notlikely due to axonal pathfinding defects. In addition, these segregationdefects can not be explained by differences in the number of RGCs in C1qKO mice. There were no significant differences in the number of cells inthe peripheral or central regions of whole mount retinas in C1q KO andcontrols (FIG. 10).

Spontaneous retinal activity has been shown to drive eye specificsegregation in several species, including mouse. Retinal waves arecomposed of bursts of action potentials that correlate the firing ofneighboring RGCs, while the firing of more distant RGC is lesscorrelated. In order to be sure that the segregation defects we measuredin C1q KOs were not secondary to effects of abnormal retinal activity inC1qKO mice, we measured retinal waves in retinas dissected from P5C1qKOs and age matched wild type controls. Multielectrode recordings ofRGC firing patterns clearly indicate that C1q-deficient mice have normalretinal waves. In both WT and C1qKO retinas, neighboring ganglion cellsare more correlated in their firing than those located at furtherdistances from one another (FIG. 11).

LGN Neurons Remain Multiply Innervated in C1q KO Mice.

The segregation of retinal axons into eye-specific territories is awell-established assay for studying developmental synapse refinement,but synapse elimination also occurs in the monocular regions of thedLGN. Electrophysiological recordings of neurons in the contralateralLGN indicate that even after the bulk of RGC axons have segregated in toeye specific territories, synapse elimination continues to occur duringthe 2 weeks after eye opening. Initially (P4-P10), dLGN neurons areinnervated by multiple (ie>10) RGC axons, but by the fourth week ofpostnatal development, each dLGN neurons receive stable inputs from 1-2RGC axons.

In order to test directly whether C1q KO fail to undergo this form ofdevelopmental synapse elimination, we studied the retinogeniculatesynapses of P26-P33 C1q KO mice in acute parasagittal brain slices. Westimulated the optic tract in small incremental intensity steps, andmeasured the amplitude of evoked responses in LGN neurons in thecontralateral region adjacent to the optic tract in C1q KOs and agematched controls (red neurons shown in FIG. 4, B). Representative tracesshown in FIG. 4A are of single LGN neurons recorded from a WT and C1q KOmouse. The peaks of the rapid inward current (AMPAR, −70 mV), and slowerdecaying outward current (NMDAR, +40 mV) represent the recruitment ofindividual axons. We found that C1q KOs remain multiply-innervated(average of 4±0.3 inputs, n=21) compared to age-matched WT controls(average of 2.2±0.2 inputs, n=30, p<0.001). Although there was nosignificant difference in the maximum amplitude of AMPA or NMDAresponses between C1q KO and WTs, the average amplitude of evokedresponses were significantly reduced in C1q KOs (data not shown). Weobserved many small amplitude responses in C1q KOs (as shown in FIG. 4A)in approximately 80% of the slices obtained from C1q KO mice. Assummarized in FIG. 11C, 81% of the cells recorded were classified asunrefined (greater than 2 inputs) compared to 27% in age matched wildtype controls (C1q KO n=21, WT n=30 cells, p<0.001).

Taken together, these experiments indicate that C1q is necessary for thestructural and functional elimination inappropriate synaptic connectionsin the developing retinogeniculate pathway.

Role of the Complement Cascade in Developmental Synapse Elimination.

C1q is the initiating protein of the classical complement cascade.Activated C1q undergoes a conformational change to activate a sequentialcascade of downstream proteases, including the major complement protein,C3. Activated C3 fragments (C3b, iC3b) can directly opsonize dead cellsand pathogens for phagocytosis, much like C1q. Alternatively,C3-activation can activate downstream complement proteins C5-C9 toeliminate unwanted cells via the formation of a membrane attack complex,which permeabilizes cell and lyses the cell.

To determine whether C3 is necessary for synapse elimination, weobtained mice deficient in complement protein C3 to investigate whetherabsence of C3 mimics the phenotype we observed in the LGNs ofC1q-deficient mice. We performed similar anterograde tracing experimentsand electrophysiological recording of retino-geniculate synapses in C3KOmice and age matched controls as described above. Consistent with ourhypothesis, C3 KO mice have defects in LGN segregation and synapseelimination that closely mimic the C1qKO phenotype. C3 KO mice hadsignificant defects in eye-specific segregation, both at P10 and P30(FIG. 5 A-C). Quantification of the percentage of dLGN occupied byoverlapping retinal axons from both eyes indicate that C3KO mice hadsignificantly more overlap than age matched and littermate controls.Electrophysiological recordings of P30-34 dLGN neurons indicate that LGNneurons recorded from C3 KO mice remain multi-innervated and had similarresponse properties to C1q KO mice (FIG. 5D). Together, these findingsprovide supporting evidence that the classical complement cascademediates normal developmental synapse elimination.

Complement C3 is Expressed at Developing CNS Synapses.

Is C3 activated and localized to synapses in the postnatal brain?Complement activation has been shown to occur in the brain after injury,stroke, and inflammation, but the expression and localization duringnormal postnatal development has not been previously examined. Weperformed immunostaining experiments in cryosections of perfusedpostnatal mouse and rat brain using polyclonal antibodies against ratC3. We found that C3 protein is expressed in postnatal rodent brain andcortex, but C3 protein was not detected in the adult brain (FIG. 6A).Importantly, double immunohistochemistry with the synaptic antibody SV2,revealed that many C3-positive puncta co-localized with synaptic puncta(FIG. 6A). Immunoreactivity was specific for C3, as C3-specificantibodies failed to stain brain sections prepared from C3-deficientmice.

Western blot analysis of protein lysates prepared from perfused,developing cortex provide direct evidence that C3 is activated in thedeveloping brain (FIG. 7B). When native C3 is cleaved, activated C3fragments (43 kD) can be detected by SDS-PAGE and western blot analysesusing polyclonal antibodies against C3. As shown in FIG. 7B, we observeda clear band for iC3b in postnatal cortex, and C3b levels weresignificantly down-regulated by P30. Together these data provideevidence that the complement cascade is activated during the period ofsynaptic pruning in the brain.

Complement-Deficient Mice have More Synapses.

An increase in synapse number would provide further evidence thatcomplement proteins are required for synapse elimination. To addressthis question, we compared the relative intensity of synaptic stainingthe LGNs and superior colliculi of C1q KO mice and littermate controlsusing several synaptic antibodies including vGLUT2, a vesicularglutamate transporter that is selectively expressed by retinal ganglioncells. As shown in FIG. 8, we found a general increase in the intensityof vGLUT2 staining in both the superior colliculi (SC) and dLGN of C1qKO mice (FIG. 8A, B). Sections from P16 C1qKO and controls wereprocessed and immunostained in parallel, and all images were collectedat identical cameral exposures. In addition, high resolution confocalmicroscopy imaging revealed a higher density of PSD95 puncta in dLGNs ofP16 C1q KO mice compared to littermate controls. (FIG. 8C).

C1q is Localized to Synapses in Early Stages of NeurodegenerativeDisease.

Could synapse loss at early stages of neurodegenerative disease involvea re-activation of developmental synapse elimination mediated by thecomplement cascade? Complement expression and activation are known to besignificantly enhanced following brain injury and in variousneurodegenerative diseases, such as Alzheimer's Disease (AD). In manycases, astrocytes, and microglia become reactive and could thereforere-express the signal that induces C1q expression in developing neurons.For example, AD is caused by a massive degeneration of synapses, and ithas become increasingly clear that synapse loss occurs long beforeneuropathology and clinical symptoms in AD brains. C1q protein is 10- to80-fold up-regulated in AD brain, and amyloid β-peptide (Aβ) is apowerful activator of C1q.

We investigated this question using a well characterized mouse model ofglaucoma. The glaucomas are neurodegenerative diseases involving deathof retinal ganglion cells and optic nerve head excavation that is oftenassociated with elevated intraocular pressure (IOP). DBA/2J mice havebeen shown to exhibit an age-related progressive glaucoma. The periodwhen mice have elevated 10P extends from 6 months to 16 months, with 8-9months representing an important transition to high 10P for many mice.Optic nerve degeneration follows IOP elevation, with the majority ofoptic nerves being severely damaged by 12 months of age.

Could C1q-dependent synapse elimination trigger synaptic loss before RGCdeath and neurodegeneration? To address this question, we looked at thetiming and localization of C1q immunoreactivity in the RGC and synapticlayers of retinas of DBA/2J and control mice at various stages ofdisease (pre-glaucoma, early, moderate and late stage). As shown in FIG.15, we found punctate C1q immunoreactivity in the IPL of retinas fromDBA/2J mice with moderate (Grade III, 6-12 months) glaucoma. At thisstage, there is no significant RGC or synapse cell loss, as indicated byPSD-95 staining in the IPL. C1q staining is present in the IPL, but lesspronounced in retinas collected from retinas with severe glaucoma(Severe, 9-12 months). At this stage there is significant RGC and deathand degeneration of retinal axons. Importantly, C1q immunoreactivity wasnot observed in the IPL of young DBA mice (1-2 months), or in retinasobtained from a control strain (2-12 months of age) that lacked theglaucoma gene. Together our findings indicate that C1q is localized tosynapses in the IPL before significant synapse loss and RGC death, whichsupports the notion that complement is mediating synapse loss in earlystages of disease.

Example 2

The decline of cognitive function is one of the greatest health threatsfor the aging population. It has been widely accepted that it isprimarily loss of synaptic connections, rather than loss of neurons perse, that is responsible for age-associated cognitive impairment inotherwise healthy individuals. In rats, these aging changes areparticularly prominent in the dentate gyrus where decreased synapses,decreased EPSPs, decreased LTP, and spatial memory deficits occur(Morrell, PNAS, 1986; Barnes, J. Comp. Physiol. Psych. 1979; Lynch, J.Gerontology, 1977).

As demonstrated in example 1 above, a vast loss of synapses occursnormally within the developing brain as neural circuits are sculpted.This developmental CNS synapse elimination is mediated by complementcomponent C1. The known role of Clq, when activated, is to trigger theonset of the classical complement cascade, causing destruction andelimination of pathogens, apoptotic cells and debris.

To investigate whether Clq plays a role in synapse loss in aging, arabbit monoclonal antibody was generated to Clq and used to examine thedetailed expression pattern for Clq protein in the adult and agingrodent CNS. Rabbits were immunized with a C1q protein purified frommouse serum. 5 hybridomas were generated, which produced 5 mAbs, all ofwhich showed identical and specific immunostaining signals (see, e.g.FIG. 16).

We discovered that Clq protein has an intriguing expression pattern inthe adult, suggesting synaptic functions for Clq and complement in thehealthy mature CNS. Clq protein levels dramatically increase in theaging brain (FIG. 17). This expression pattern is detected in C57BL/6,Swiss Webster, DBA/2, BALB/c mouse (FIG. 18A, 18B) and in both male(FIG. 18A) and female (FIG. 18B) tissue, indicating that it is strainand sex independent. The most dramatic increase can be observed bycomparing a postnatal day 6 (P6) mouse brain (FIG. 21) with that of a 24month old mouse (FIG. 22).

Western blots of C1q levels in brain homogenates likewise demonstrate adramatic increase of C1q protein as the brain ages (FIGS. 23 and 24).C1q protein levels do not increase in aging mouse serum (FIG. 25).Consistent with this, an analysis of perfused and non-perfused tissuefrom differently aged mice indicates that the increase in CNS C1qprotein levels cannot be explained by an age-dependent leakage of theBlood-Brain Barrier (FIG. 26).

It was observed that C1q immunoreactivity forms patches in the adultbrain (FIG. 22). These patches represent punctate signals of 50 to 200uM with stronger C1q intensity compared to signals in the surroundingneuropil. They are most pronounced in cortex, thalamus, and midbrain,but, unlike amyloid plaques, are rare in hippocampus. They arethioflavin negative, indicating that they are not associated with highermicroglial density or synapse loss. They can be detected in youngadulthood, however, are more pronounced in middle-aged (6 months onward)and even more in aged mouse brain.

To determine which cells expressed C1q, in situ hybridization withprobes for C1q-A and CXCR1 was performed. At P30, most or all microgliaexpress C1q (FIG. 27). C1q immunoreactivity is also found in a subset ofneurons in the developing and adult mouse brain, e.g. the hippocampus,thalamus, striatum, and midbrain (FIG. 28). Co-staining withparvalbumin, a marker of inhibitory neurons, indicates that C1qimmunoreactive neurons are mainly inhibitory (FIG. 29).

C1q immunostaining also indicates that the increase in C1qimmunoreactivity with aging is highly synaptically localized, appears atP15 coincident with synapse formation, and is especially high in thedentate gyrus (FIG. 32). Synaptic localization was confirmed byco-staining with synaptophysin (FIG. 34). Co-staining with VGluT1indicated that C1q immunreactivity localizes to many excitatory synapsesin the dentate gyrus of older mice (FIG. 35c ), while co-staining withGephryn indicated that C1q immunreactivity localizes to many inhibitorysynapses in the stratum lacunosum (FIG. 35d ). Electron microscopyrevealed that C1q could be found in close proximity to synapses (FIG.35e-f ).

To determine if the classical complement cascade is activate duringnormal aging, the expression levels of C3 were assessed in differentaged mice. C3 protein levels were high shortly after birth (P6), asexpected in view of the extensive remodeling and synapse eliminationthat occurs during this developmental window (FIGS. 19, 20, P6).Interestingly, measurable C3 protein levels were also observed throughadulthood, indicating that the classical complement cascade is active inthe CNS throughout adult life.

In summary, C1q expression in the CNS is developmentally regulated, C1qlevels being low at birth and increasing during maturation and aging.C1q is expressed by microglia throughout the brain and by a largepercentage (but not all) inhibitory neurons. In some embodiments, theC1q inhibitor targets inhibitory neurons. The increase in C1q proteinwith aging primarily occurs by accumulation at synapses throughout theCNS starting in hippocampus (dentate gyrus), cortex, midbrain, followedby thalamus, brainstem, and finally cerebellum. These findings supportthe conclusions above that increased activation of the complementcascade in aging and other age-related neurodegenerative conditionscauses excessive synapse loss which results in cognitive decline.

Example 3

Wild type mice and C1q-deficient mice were assessed using severalelectrophysiological tests to determine the state of decline in functionof hippocampal neurons at 3 months of age, 9 months of age, and 15months of age. This included assessing the Perforant Path/Dentate Gyrussynaptic pathway for input output curves of dentate gyrus molecularlayer field potentials, which measure the relationship between stimulussize and the size of the evoked synaptic response to address synapticproperties or architecture; the Perforant Path/Dentate Gyrus pathway forpaired-pulse facilitation, which measures the rapid succession of twopresynaptic stimuli; and the Perforant Path/Dentate Gyrus pathway andSchaffer collateral/CA1 pathway for long-term potentiation, whichmeasures the extent of long-lasting enhancement in signal transmissionbetween two neurons that results from synchronus, high-frequencystimulation.

In addition, wild type mice and C1q-deficient mice were assessed usingthe following behavior tests to determine the state of cognitive declineat 3 months of age, 9 months of age, and 15 months of age:

-   -   SHIRPA: systematically assess the general health and behavioral        traits of a transgenic mouse.    -   Activity Chamber: determine general activity levels, gross        locomotion activity, and exploration habits.    -   Open Field: measure behavioral responses such as locomotor        activity, hyperactivity, exploratory behaviors, anxiety.    -   Y-Maze: Hippocampus-dependent navigation behaviors and spatial        working memory.    -   Novel Object Recognition: Hippocampus and pre-frontal        cortex-dependent spatial displacement and recognition memory.    -   Morris Water Maze: Hippocampus-dependent spatial learning and        memory Contextual and tone-cued Fear Conditioning:        Hippocampus/Amygdala-dependent Pavlovian (associative) learning.

Adult C1q deficient mice were healthy overall and exhibited normalbehavior in basic behavioral tasks as well as in fear conditioning/fearextinction analysis (FIG. 40). The number and morphology of dendriticspines was comparable to that of wild type mice (FIG. 36). However, asdemonstrated in FIGS. 37-38, less aging-associated decline in neuronalfunction was observed in C1q deficient mice as compared to wild typemice both by electrophysiology measures and by behavioral studies.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which will be limited only by the appendedclaims.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. All technicaland scientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs unless clearly indicated otherwise.

What is claimed is:
 1. A method of treating a patient suffering fromcognitive decline, the method comprising administering to the patient anantibody that binds to C1q, C1s, or C1r.
 2. The method of claim 1,wherein the antibody binds to C1q.
 3. The method of claim 1, wherein theantibody binds to C1s.
 4. The method of claim 1, wherein the antibodybinds to C1r.
 5. The method of claim 1, wherein the cognitive decline istriggered or exacerbated by synapse loss.
 6. The method of claim 1,wherein the cognitive decline is triggered or exacerbated by injury tothe brain.
 7. The method of claim 6, wherein the injury to the brain issepsis, trauma, toxin, or ischemia.
 8. The method of claim 1, whereinthe cognitive decline is a dementia or memory loss.
 9. The method ofclaim 1, wherein the cognitive decline is age-associated cognitiveimpairment.
 10. The method of claim 1, wherein the cognitive decline isa worsening of mental functions over time.
 11. The method of claim 10,wherein the worsening of mental functions is an inability to plan orfollow instructions.
 12. A method of inhibiting synapse loss in apatient suffering from cognitive decline, the method comprisingadministering to the patient an antibody that binds to C1q, C1s, or C1r.13. The method of claim 12, wherein the antibody binds to C1q.
 14. Themethod of claim 12, wherein the antibody binds to C1s.
 15. The method ofclaim 12, wherein the antibody binds to C1r.
 16. The method of claim 12,wherein the cognitive decline is triggered or exacerbated by injury tothe brain.
 17. The method of claim 16, wherein the injury to the brainis sepsis, trauma, toxin, or ischemia.
 18. The method of claim 12,wherein the cognitive decline is a dementia or memory loss.
 19. Themethod of claim 12, wherein the cognitive decline is age-associatedcognitive impairment or mild cognitive impairment.
 20. The method ofclaim 12, wherein the cognitive decline is a worsening of mentalfunctions.
 21. The method of claim 20, wherein the worsening of mentalfunctions is an inability to plan or follow instructions.